Globin gene therapy for treating hemoglobinopathies

ABSTRACT

The presently disclosed subject matter provides for expression cassettes that allow for expression of a globin gene or a functional portion thereof, vectors comprising thereof, and cells transduced with such expression cassettes and vectors. The presently disclosed subject matter further provides methods for treating a hemoglobinopathy in a subject comprising administering an effective amount of such transduced cells to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US15/48698 filed Sep. 4, 2015, which claims priority to U.S.Provisional Application No. 62/045,997 filed Sep. 4, 2014, the contentsof which is hereby incorporated by reference in its entirety herein, andto each of which priority is claimed.

GRANT INFORMATION

This invention was made with government support under Grant No. HL053750from National Heart, Lung and Blood Institute. The government hascertain rights in the invention.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted via EFS on Mar. 3, 2017. Pursuant to 37 C.F.R. §1.52(e)(5),the Sequence Listing text file, identified as 0727340477SL, is 126,316bytes in size and was created on Mar. 3, 2017. The Sequence Listing,electronically filed on Mar. 3, 2017, does not extend beyond the scopeof the specification and thus does not contain new matter.

INTRODUCTION

The presently disclosed subject matter provides expression cassettes andvectors comprising such expression cassettes that express a globinprotein, e.g., a human β-globin protein. The presently disclosed subjectmatter further provides expression cassettes that comprise a globin geneor a functional portion thereof operably linked to a β-globin locuscontrol region (LCR) comprising a plurality of Dnase I hypersensitivesites. The expression cassettes of the presently disclosed subjectmatter comprise one or more insulators that counteract the effect ofenhancer elements. The insulators disclosed herein do not substantiallyadversely impact the titer of a vector that comprises the presentlydisclosed expression cassettes. The expression cassettes and vectors canbe used for treating a hemoglobinopathy, e.g., β-thalassemia, and sicklecell anemia.

BACKGROUND

β-thalassemia and sickle cell anemia are severe congenital anemias thatare caused by defective production of the β chain of hemoglobin. Inβ-thalassemia, the β chain deficit leads to the intracellularprecipitation of excess α-globin chains, causing ineffectiveerythropoiesis and hemolytic anemia (Weatherall and Clegg (1981),Stamatoyannopoulos et al., (1994), Weatherall (2001), Steinberg (2001)).In the most severe forms found in homozygotes or compound heterozygotes,anemia is lethal within the first years of life in the absence of anytreatment (Cooley and Lee (1925)). Lifelong transfusion therapy isneeded to correct anemia, suppress ineffective erythropoiesis andinhibit gastrointestinal iron absorption (Weatherall and Clegg (1981),Stamatoyannopoulos et al. (1994), Weatherall (2001), Steinberg (2001)).However, transfusion therapy itself leads to iron overload, which islethal if untreated. The prevention and treatment of iron overload arethe major goals of current patient management (Giardina (2001)). Theonly current curative treatment to cure β-thalassemia is to provideerythroid precursors harboring normal globin genes through allogeneicbone marrow transplantation (BMT) (Giardini and Lucarelli (1994), Bouladet al. (1998), Lucarelli et al. (1999), Tisdale and Sadelain (2001)).

In sickle cell anemia, the hemoglobin β chain is mutated at amino acidposition 6 (Glu→Val), leading to the synthesis of β^(S) instead of thenormal β^(A) chain (Steinberg (2001), Pauling et al. (1949)). Theresulting hemoglobin, HbS, causes accelerated red cell destruction,erythroid hyperplasia and painful vaso-occlusive ‘crises’ (Steinberg(2001)). Vaso-occlusion can damage organs, eventually causing long-termdisabilities (e.g. following stroke or bone necrosis), and sometimessudden death. While a very serious disorder, the course of sickle celldisease is typically unpredictable (Steinberg (2001)). By increasingproduction of fetal hemoglobin (Swank and Stamatoyannopoulos (1998)) andsuppressing hematopoiesis, hydroxyurea can produce a measurable clinicalbenefit (Platt et al. (1984)), Charache et al. (1992), Atweh andLoukopoulos (2001)). Since hydroxyurea is a cytotoxic agent, there is agreat need for alternative, less toxic drugs to induce γ-globin geneexpression (Perrine et al. (2005), Stamatoyannopoulos (2005)). As forβ-thalassemia, allogeneic bone marrow transplantation (BMT) is atpresent the only curative therapy for sickle cell disease (Tisdale andSadelain (2001), Vermylen et al. (1998), Luzzatto and Goodfellow(1989)).

BMT, however, is not available as a therapeutic option to most patientssuffering from β-thalassemia or sickle cell disease, due to the lack ofan HLA-matched bone marrow donor for most individuals. Furthermore,although potentially curative, allogeneic BMT is not devoid ofcomplications. Safe transplantation requires the identification of ahisto-compatible donor to minimize the risks of graft rejection andgraft-versus-host disease (Tisdale and Sadelain (2001), Vermylen et al.(1998), Luzzatto and Goodfellow (1989)). Because of the greater risksassociated with matched-unrelated or mismatched transplants, mostpatients have to settle for life-long transfusion therapy, which doesnot correct ineffective erythropoiesis and exacerbates systemic ironaccumulation. Moreover, despite the considerable improvement in lifeexpectancy in the last decades (Borgna-Pignatti et al. (2004), Telfer etal. (2009), Ladis et al. (2011)), the risk of some serious complicationsarising over the long term from viral infections, iron toxicity andliver cirrhosis, remain (Mancuso et al. (2006)). These medical risks,together with the socio-economic cost of chronic β-thalassemia,underscore the need for safe, effective and curative therapies.

The only means to cure rather than treat severe β-thalassemia is toprovide the patient with healthy hematopoietic stem cells (HSCs). HSCsnormally give rise to all blood cell types, including 20 billion RBCsper day in adults. HSCs can be harvested from a donor with wild-typeβ-globin genes to yield long-lived red blood cells (RBCs) with a normalcontent in hemoglobin. Alternatively, one may genetically correct thepatient's own HSCs, which at once resolves the search for a donor andeliminates the risks of graft-versus-host disease and graft rejectionassociated with allogeneic BMT (Sadelain (1997), Sadelain et al.(2007)). Globin gene transfer aims to restore the capacity of theβ-thalassemic subject's own blood-forming stem cells to generate RBCswith a sufficient hemoglobin content Sadelain et al. (2007), Persons andTisdale (2004), Sadelain (2006)). The goal in patients with sickle cellanemia is to prevent sickling, which can be achieved by diluting theendogenous HbS with a non-sickling Hb that incorporates thevector-encoded globin chain. The patient's own HSCs are the cells thathave to be genetically modified to ensure long-lasting therapeuticbenefits and achieve a curative stem cell-based therapy.

The implementation of globin gene transfer for the treatment of severeβ-thalassemia and sickle cell anemia requires the efficient introductionof a regulated human β- or β-like globin gene in HSCs. The β-globin gene(or β-like variant) must be expressed in erythroid-specific fashion andat high level, especially for the treatment of transfusion-dependentbeta-zero thalassemias.

The globin vectors developed to date present shortcomings that may limitor even preclude their safe use in thalassemia and sickle cell patients.Some of the β-globin locus control region (LCR) components contained inthe vectors, in particular Dnase I hypersensitive site-2 (HS2), may havenon-erythroid activity, exposing patients to the risk of insertionaloncogenesis as seen with non-specific expression vectors. Further, theuse of large LCR segments can be detrimental to the production of hightiter vectors and the efficient transduction of patients HSCs.Accordingly, there is a need for novel globin expression cassettes thatallow for therapeutic expression of a globin gene (e.g., human β-globingene) in erythroid-specific and differentiation stage-specific fashionwith minimal risk of insertional oncogenesis, and that enable high leveltransduction, thus improving their safety when used in treatingthalassemia and sickle cell patients.

SUMMARY OF THE INVENTION

The presently disclosed subject matter generally provides enhancerblocking insulators, and certain insulators additionally possess barrierinsulator activity. The presently disclosed subject matter also providesexpression cassettes comprising one or more insulators and allows forexpression of a globin gene (e.g., a human β globin gene). Also providedare vectors comprising such expression cassettes, cells transduced withsuch expression cassettes or such vectors, and uses of such expressioncassettes for treating hemoglobinopathies (e.g., β-thalassemia andsickle cell anemia).

In certain non-limiting embodiments, the presently disclosed subjectmatter provides an insulator comprising the CTCF binding site sequenceset forth in SEQ ID NO:18, for example, but not limited to, an insulatorcomprising SEQ ID NO: 24 or SEQ ID NO:25, such as an insulator havingthe nucleotide sequence set forth in SEQ ID NO:1 (and see infra). Thepresently disclosed subject matter also provides expression cassettescomprising at least one insulator comprising the CTCF binding sitesequence set forth in SEQ ID NO:18, for example, but not limited to, aninsulator comprising SEQ ID NO: 24 or SEQ ID NO:25, such as an insulatorhaving the nucleotide sequence set forth in SEQ ID NO:1. In anon-limiting embodiment, an expression cassette comprises at least oneinsulator comprising the CTCF binding site sequence set forth in SEQ IDNO:18, for example, but not limited to, an insulator comprising SEQ IDNO: 24 or SEQ ID NO:25, such as an insulator having the nucleotidesequence set forth in SEQ ID NO:1, and a globin gene or a functionalportion thereof operably linked to a β-globin locus control region(LCR). In certain embodiments, the β-globin LCR does not comprise aDnase I hypersensitive site-2 (HS2) region. In certain embodiments, theβ-globin LCR region does not comprise a core sequence of HS2. In onenon-limiting embodiment, the core sequence of HS2 has the nucleotidesequence set forth in SEQ ID NO:20. In one non-limiting embodiment, thecore sequence of HS2 has the nucleotide sequence set forth in SEQ IDNO:21. In certain embodiments, the β-globin LCR does not comprise a HS2region that sustains the enhancer activity of HS2. In one non-limitingembodiment, the β-globin LCR comprises a Dnase I hypersensitive site-1(HS1) region, a Dnase I hypersensitive site-3 (HS3) region, and a DnaseI hypersensitive site-4 (HS4) region. In certain embodiments, the HS3region is positioned between the HS1 and the HS4 region.

In certain embodiments, the HS1 region is about 1.1 kb bp in length. Inone non-limiting embodiment, the HS1 region is between about 500 bp andabout 1000 bp in length. In one non-limiting embodiment, the HS1 regionhas the nucleotide sequence set forth in SEQ ID NO:2. In certainembodiments, the HS1 region is about 600 bp in length. In onenon-limiting embodiment, the HS1 region is 602 bp in length. In certainembodiments, the HS1 region is between about 500 and about 600 bp inlength. In one non-limiting embodiment, the HS1 region has thenucleotide sequence set forth in SEQ ID NO:3. In certain embodiments,the HS1 region is about 490 bp in length. In one non-limitingembodiment, the HS1 region is 489 bp in length. In one non-limitingembodiment, the HS1 region has the nucleotide sequence set forth in SEQID NO:4. In one non-limiting embodiment, the β-globin LCR comprises aHS1 region having a nucleotide sequence set forth in SEQ ID NO:2, a HS3region having a nucleotide sequence set forth in SEQ ID NO:5, and a HS4region having a nucleotide sequence set forth in SEQ ID NO:6, and theβ-globin LCR region does not comprise a HS2 region. In one non-limitingembodiment, the β-globin LCR region comprises a HS1 region having anucleotide sequence set forth in SEQ ID NO:3, a HS3 region having anucleotide sequence set forth in SEQ ID NO:5, and a HS4 region having anucleotide sequence set forth in SEQ ID NO:8, and the β-globin LCR doesnot comprise a HS2 region. In one non-limiting embodiment, the β-globinLCR comprises a HS1 region having a nucleotide sequence set forth in SEQID NO:4, a HS3 region having a nucleotide sequence set forth in SEQ IDNO:5, and a HS4 region having a nucleotide sequence set forth in SEQ IDNO:8, and the β-globin LCR does not comprise a HS2 region.

In certain embodiments, the β-globin LCR region does not comprise a HS1region and/or does not comprise a HS2 region, and the β-globin LCR doesnot comprise a core sequence of HS2. In certain embodiments, theβ-globin LCR does not comprise a core sequence of HS1. In onenon-limiting embodiment, the core sequence of HS1 has the nucleotidesequence set forth in SEQ ID NO:22. In one non-limiting embodiment, thecore sequence of HS1 has the nucleotide sequence set forth in SEQ IDNO:23. In certain embodiments, the β-globin LCR does not comprise a HS1region that sustains the function of HS1. In certain embodiments, theβ-globin LCR comprises a HS3 region and a HS4 region and does notcomprise a core sequence of HS1. In certain embodiments, the HS3 regionis positioned between a globin gene or functional portion thereof andthe HS4 region. In certain embodiments, the HS3 region is between about200 and about 1400 bp in length, e.g., between about 1300 and 1400 bp inlength. In certain embodiments, the HS3 region is about 1300 bp inlength. In one non-limiting embodiment, the HS3 region is 1301 bp inlength. In one non-limiting embodiment, the HS3 region has thenucleotide sequence set forth in SEQ ID NO:5. In certain embodiments,the HS4 region is between about 200 and about 1200 bp in length, e.g.,between about 400 and 1100 bp in length. In certain embodiments, the HS4region is about 1.1 kb in length. In one non-limiting embodiment, theHS4 region is 1065 bp in length. In one non-limiting embodiment, the HS4region has the nucleotide sequence set forth in SEQ ID NO:6. In onenon-limiting embodiment, the HS4 region has the nucleotide sequence setforth in SEQ ID NO:7. In certain embodiments, the HS4 region is about450 bp in length. In one non-limiting embodiment, the HS4 region is 446bp in length. In one non-limiting embodiment, the HS4 region has thenucleotide sequence set forth in SEQ ID NO:8. In one non-limitingembodiment, the β-globin LCR region comprises a HS3 region having thenucleotide sequence set forth in SEQ ID NO:5 and a HS4 region having anucleotide sequence set forth in SEQ ID NO:6, and the β-globin LCRregion does not comprise a HS1 region or a HS2 region.

Alternatively, the β-globin LCR region can comprise a HS2 region, a HS3region, and a HS4 region. In certain embodiments, the HS2 region isbetween about 400 and about 1000 bp in length, e.g., between about 800and 900 bp in length. In certain embodiments, the HS2 region is about860 bp in length. In one non-limiting embodiment, the HS2 region has thenucleotide sequence set forth in SEQ ID NO:9. In certain embodiments,the HS3 region is about 1300 bp in length. In one non-limitingembodiment, the HS3 region is 1301 bp in length. In one non-limitingembodiment, the HS3 region has the nucleotide sequence set forth in SEQID NO:5. In certain embodiments, the HS4 region is about 1.1 kb inlength. In one non-limiting embodiment, the HS4 region is 1065 bp inlength. In one non-limiting embodiment, the HS4 region has thenucleotide sequence set forth in SEQ ID NO:7. In one non-limitingembodiment, the β-globin LCR region comprises a HS2 region having thenucleotide sequence set forth in SEQ ID NO:9, a HS3 region having thenucleotide sequence set forth in SEQ ID NO:5, and a HS4 region havingthe nucleotide sequence set forth in SEQ ID NO:7. Additionally, theβ-globin LCR region can further comprise a HS1 region.

In certain embodiments, the globin gene is selected from the groupconsisting of β-globin gene, γ-globin gene, and δ-globin gene. In onenon-limiting embodiment, the globin gene is human β-globin gene. Innon-limiting embodiments, the human β-globin gene is selected from thegroup consisting of a wild-type human β-globin gene, a deleted humanβ-globin gene comprising one or more deletions of intron sequences, anda mutated human β-globin gene encoding at least one anti-sickling aminoacid residue. In one non-limiting embodiment, the human β-globin gene ishuman β^(A)-globin gene encoding a threonine to glutamine mutation atcodon 87 (β^(A-T87Q)).

In certain embodiments, the expression cassette comprises one insulatorcomprising the CTCF binding site sequence set forth in SEQ ID NO:18, forexample, but not limited to, an insulator comprising SEQ ID NO: 24 orSEQ ID NO:25, such as an insulator having the nucleotide sequence setforth in SEQ ID NO:1. In certain embodiments, the expression cassettecomprises two insulators, each comprising the CTCF binding site sequenceset forth in SEQ ID NO:18, for example, but not limited to, where one orboth insulators comprise SEQ ID NO: 24 or SEQ ID NO:25 and/or have thenucleotide sequence set forth in SEQ ID NO:1.

In certain embodiments, the expression cassette further comprises aβ-globin promoter. In certain embodiments, the β-globin promoter ispositioned between the globin gene or functional portion thereof andβ-globin LCR region. In certain embodiments, the β-globin promoter isbetween about 200 and about 700 bp in length. In one non-limitingembodiment, the 3-globin promoter is a human β-globin promoter that isabout 613 bp in length. In one non-limiting embodiment, the humanβ-globin promoter has the nucleotide sequence set forth in SEQ ID NO:10.In another non-limiting embodiment, the β-globin promoter is a humanβ-globin promoter that is about 265 bp in length. In one non-limitingembodiment, the β human β-globin promoter has the nucleotide sequenceset forth in SEQ ID NO:11.

In certain embodiments, the expression cassette further comprises ahuman β-globin 3′ enhancer. In certain embodiments, the human β-globin3′ enhancer is positioned in the upstream of the globin gene orfunctional portion thereof. In certain embodiments, the β-globin 3′enhancer is between about 700 and about 900 bp in length, e.g., betweenabout 800 and 900 bp in length. In one non-limiting embodiment, thehuman β-globin 3′ enhancer is about 879 bp in length. In onenon-limiting embodiment, the human β-globin 3′ enhancer has thenucleotide sequence set forth in SEQ ID NO:12.

In certain embodiments, the expression cassette further comprises atleast one erythroid-specific enhancer. In certain embodiments, the atleast one erythroid-specific enhancer is positioned between the globingene or functional portion thereof and the β-globin LCR region. Incertain embodiments, the at least one erythroid-specific enhancer has anucleotide sequence selected from the group consisting of SEQ ID NOS:13, 14, 15, 16 and 17. In certain embodiments, the at least oneerythroid-specific enhancer is between about 100 and about 200 bp inlength. In certain embodiments, the expression cassette comprises one,two or three erythroid-specific enhancers.

In certain embodiments, the expression cassette allows for expression ofthe globin gene or functional portion thereof in a mammal. In onenon-limiting embodiment, the expression cassette allows for expressionof a human β-globin gene. In certain embodiments, the expression of theglobin gene or functional portion thereof is restricted to erythroidtissue.

The presently disclosed subject matter also provides recombinant vectorscomprising the above-described expression cassettes. In certainembodiments, the recombinant vector is a retroviral vector. In onenon-limiting embodiment, the retroviral vector is a lentivirus vector.In certain embodiments, the expression cassette comprised in therecombinant vector comprises one insulator. In certain embodiments, therecombinant vector further comprises a Woodchuck hepatitispost-regulatory element (WPRE) in the 3′ long terminal repeat (LTR) ofthe vector. In certain embodiments, the recombinant vector furthercomprises a bovine growth hormone polyadenylation signal in the 3′ longterminal repeat (LTR) of the vector.

In addition, the presently disclosed subject matter providesnon-naturally occurring or engineered nucleases comprising theabove-described expression cassettes. In certain embodiments, thenuclease is selected from the group consisting of a non-naturallyoccurring or engineered zinc-finger nuclease (ZFN), a non-naturallyoccurring or engineered meganuclease, and a non-naturally occurring orengineered transcription activator-like effector nuclease (TALEN). Incertain embodiments, the nuclease comprises a DNA binding domain and anuclease cleavage domain. In certain embodiments, the nuclease binds toa genomic safe harbor site. In certain embodiments, the nucleasegenerates a double strand break (DSB) at the genomic safe harbor site.In certain embodiments, the expression cassette comprised in thenuclease comprises two of the insulator having the nucleotide sequenceset forth in SEQ ID NO:1. In certain embodiments, the nuclease allowsfor targeted delivery of the expression cassette. The presentlydisclosed subject matter also provides polynucleotides encoding theabove-described nucleases, and vectors comprising the polynucleotides.In one non-limiting embodiment, the vector is a lentiviral vector.

Furthermore, the presently disclosed subject matter providesnon-naturally occurring or engineered CRISPR-Cas systems comprising theabove-described expression cassettes. In certain embodiments, theCRISPR-Cas system comprises a CRISPR-Cas nuclease and single-guide RNA.In certain embodiments, the CRISPR-Cas system binds to a genomic safeharbor site. In certain embodiments, the CRISPR-Cas system generates adouble strand break (DSB) at the genomic safe harbor site. In certainembodiments, the expression cassette comprised in the CRISPR-Cas systemcomprises two of the insulator having the nucleotide sequence set forthin SEQ ID NO:1. In certain embodiments, the CRISPR-Cas allows fortargeted delivery of the expression cassette. The presently disclosedsubject matter also provides polynucleotides encoding theabove-described CRISPR-Cas systems, and vectors comprising thepolynucleotides. In one non-limiting embodiment, the vector is alentiviral vector.

In some embodiments, the genomic safe harbor site is an extragenicgenomic safe harbor site. In certain embodiments, the genomic safeharbor site is located on chromosome 1. In some embodiments, the genomicsafe harbor meets all of the following five criteria: (1) distance of atleast 50 kb from the 5′ end of any gene (e.g., from the 5′ end of thegene), (ii) distance of at least 300 kb from any cancer-related gene,(iii) within an open/accessible chromatin structure (measured by DNAcleavage with natural or engineered nucleases), (iv) location outside agene transcription unit and (v) location outside ultraconserved regions(UCRs), microRNA or long non-coding RNA of the human genome.

Additionally, the presently disclosed subject matter provides cellstransduced with the above-described expression cassettes, cellstransduced with the above-described recombinant vectors, cellstransduced with the above-described nucleases, cells transduced with theabove-described CRISPR-Cas systems. In addition, the presently disclosedsubject matter provides cells transduced with the above-describedvectors. In certain embodiments, the cell is selected from the groupconsisting of a hematopoietic stem cell, an embryonic stem cell, aninduced pluripotent stem cell, and a hemogenic endothelium cell. In onenon-limiting embodiment, the hematopoietic stem cell is a CD34⁺hematopoietic stem cell. In certain embodiments, the cell is transducedex vivo.

Also provided are pharmaceutical compositions comprising an effectiveamount of the above-described cells and a pharmaceutically acceptablecarrier. The presently disclosed subject matter also providespharmaceutical compositions for treating a hemoglobinopathy comprisingan effective amount of the above-described cells and a pharmaceuticallyacceptable carrier.

Furthermore, the presently disclosed subject matter provides kits fortreating a hemoglobinopathy comprising the above-described cells. Incertain embodiments, the kits further comprise written instructions forusing the cell for treating a subject having a hemoglobinopathy.

In addition, the presently disclosed subject matter provides methods oftreating a hemoglobinopathy in a subject, comprising administering aneffective amount of the above-described cells to the subject, therebyrestoring the subject's ability to produce red blood cells containingnormal hemoglobin. In certain embodiments, a therapeutically relevantlevel of hemoglobin is produced in the subject following administeringthe cell to the subject. In certain amendments, the method comprisesadministering an effective amount of the cell transduced with theabove-described recombinant vector. In some embodiments, the vector copynumber of the recombinant vector in the cell that provides for thetherapeutically relevant level of hemoglobin in the subject is about0.5-2 vector copy number per cell. In certain embodiments, the methodcorrects ineffective erythropoiesis in the subject. In certainembodiments, the method does not incur the risk of graft-versus-hostdisease in the subject. In certain embodiments, the method does notcomprise administering an immunosuppressive agent. In certainembodiments, the cell is selected from the group consisting of ahematopoietic stem cell, an embryonic stem cell, an induced pluripotentstem cell, and a hemogenic endothelium cell. In one non-limitingembodiment, the subject is a human. In certain embodiments, the cell isfrom the subject. In one non-limiting embodiment, the cell is from bonemarrow of the subject.

In accordance with the presently disclosed subject matter, thehemoglobinopathy can be selected from the group consisting of hemoglobinC disease, hemoglobin sickle cell disease (SCD), sickle cell anemia,hereditary anemia, thalassemia, β-thalassemia, thalassemia major,thalassemia intermedia, α-thalassemia, and hemoglobin H disease. In onenon-limiting embodiment, the hemoglobinopathy is β-thalassemia. Inanother non-limiting embodiment, the hemoglobinopathy is sickle cellanemia.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings.

FIG. 1 depicts a recombinant vector comprising an expression cassette inaccordance with one non-limiting embodiment of the presently disclosedsubject matter.

FIG. 2 depicts a recombinant vector an expression cassette in accordancewith one non-limiting embodiment of the presently disclosed subjectmatter.

FIG. 3 depicts a recombinant vector an expression cassette in accordancewith one non-limiting embodiment of the presently disclosed subjectmatter.

FIG. 4 depicts a recombinant vector an expression cassette in accordancewith one non-limiting embodiment of the presently disclosed subjectmatter.

FIGS. 5A-C represent the genotoxicity of insulator A1. (A) demonstratethe gammaretroviral vector genotoxicity assay used. (B) notice theincreased survival of mice receiving 32D cells transduced with insulatedgammaretroviral vector. Also notice the results obtained with cHS4 andwith the uninsulated control. (C) show that insulator A1 decreased therisk of genotoxicity.

FIG. 6 represents normalized β chain expression in thalassemicHbb^(th3/+) mice 8 and 44 weeks post-treatment.

FIG. 7 represents the evaluation of enhancer activity in non-erythroidK562 cells.

FIG. 8 represents the erythroid-specific enhancers in accordance withcertain embodiments of the presently disclosed subject matter.

FIG. 9 represents the erythroid-specific enhancers in accordance withcertain embodiments of the presently disclosed subject matter.

FIGS. 10A-B depict various recombinant vectors comprising the presentlydisclosed expression cassettes.

FIG. 11 represents the titer of the recombinant vectors comprising thepresently disclosed expression cassettes.

FIG. 12 represents the titer of the recombinant vectors comprising thepresently disclosed expression cassettes.

DETAILED DESCRIPTION OF THE INVENTION

The presently disclosed subject matter generally provides expressioncassettes that allow for expression of a globin gene (e.g., humanβ-globin gene). In one non-limiting example, the expression cassettecomprises at least one insulator comprising the CTCF binding sitesequence set forth in SEQ ID NO:18, for example, but not limited to, aninsulator comprising SEQ ID NO: 24 or SEQ ID NO:25, such as an insulatorhaving the nucleotide sequence set forth in SEQ ID NO:1 and a globingene or a functional portion thereof operably linked to a β-globin locuscontrol region (LCR) region. The expression of the globin gene inducedby the presently disclosed expression cassettes is erythroid-specific,differentiation stage-specific, high-level, and sustained. The presentlydisclosed subject matter also provides recombinant vectors,non-naturally occurring or engineered nucleases, and non-naturallyoccurring or engineered CRISPR-Cas systems comprising such expressioncassettes, and cells transduced with such expression cassettes,recombinant vectors, nucleases and CRISPR-Cas systems. The presentlydisclosed expression cassettes and vectors comprising thereof providefor a safe gene transfer therapy as therapeutic transgene expression isachieved (e.g., a therapeutically relevant level of hemoglobin isproduced) with a low vector copy number per cell (e.g., 0.5-2, 1-2, oreven 0.5-1). In addition, the presently disclosed subject matterprovides methods of using such transduced cells for treating ahemoglobinopathy (e.g., β-thalassemia and sickle cell anemia).

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

As used herein, the term “expression cassette” refers to a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements, which permit transcription of aparticular nucleic acid in a target cell. The expression cassette can beincorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA,virus or nucleic acid region. The expression cassette portion caninclude a gene to be transcribed and elements that control theexpression of the gene (e.g., a promoter).

As used herein, the term “β-globin locus control region (LCR) region”refers to a polynucleotide composed of one or more Dnase Ihypersensitive site (HS) regions, including a HS1 region, a HS2 region,a HS3 region, and a HS4 region. The structure of many LCRs of theβ-globin genes have been published, e.g., human (Li et al., J. Biol.Chem. (1985); 260:14,901; Li et al., Proc. Natl. Acad. Sci. (1990)87:8207); mouse (Shehee et al., J. Mol. Biol. (1989); 205:41); rabbit(Margot et al., J. Mol. Biol. (1989); 205:15); and goat (Li, Q., et al.,Genomics (1991); 9:488), each of which are incorporated by referenceherein. In certain embodiments, the 3-globin LCR region comprises a HS2region (e.g., a β-globin LCR region comprising a HS2 region, a HS3region and a HS4 region; and a β-globin LCR region comprising a HS1region, a HS2 region, a HS3 region and a HS4 region). In certainembodiments, the β-globin LCR region does not comprise a HS2 region(e.g., a β-globin LCR region comprising a HS1 region, a HS3 region, aHS4 region). In certain embodiments, the β-globin LCR region does notcomprise a HS2 region or a HS1 region (e.g., a β-globin LCR regioncomprising a HS3 region and a HS4 region).

As used herein, the term “recombinant” includes reference to a cell orvector, that has been modified by the introduction of a heterologousnucleic acid or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention or may have reduced or eliminated expression of a nativegene.

As used herein, the term “globin” refers to a family of heme-containingproteins that are involved in the binding and transport of oxygen.Subunits of vertebrate and invertebrate hemoglobins, vertebrate andinvertebrate myoglobins or mutants thereof are included by the termglobin.

As used herein, the term “wild-type” refers to the normal gene, virus,or organism found in nature without any mutation or modification.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene region, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA(siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide can comprise one ormore modified nucleotides, such as methylated nucleotides and nucleotideanalogs. In particular embodiments, the presently disclosed subjectmatter provides polynucleotides encoding one or more globin genes orfuctional portions thereof. If present, modifications to the nucleotidestructure may be imparted before or after assembly of the polymer. Thesequence of nucleotides may be interrupted by non nucleotide components.A polynucleotide may be further modified after polymerization, such asby conjugation with a labeling component. Such polynucleotides need notbe 100% identical with an endogenous nucleic acid sequence, but willtypically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capableof hybridizing with at least one strand of a double-stranded nucleicacid molecule. By “hybridize” is meant pair to form a double-strandedmolecule between complementary polynucleotide sequences (e.g., a genedescribed herein), or portions thereof, under various conditions ofstringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) MethodsEnzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues and tovariants and synthetic analogues of the same. Thus, these terms apply toamino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers. Particular embodiments ofthe presently disclosed subject matter also include polypeptide“variants.” Polypeptide “variant” refers to polypeptides that aredistinguished from a reference polypeptide by the addition, deletion,truncations, and/or substitution of at least one amino acid residue, andthat retain a biological activity. In certain embodiments, a polypeptidevariant is distinguished from a reference polypeptide by one or moresubstitutions, which may be conservative or non-conservative, as knownin the art. In certain embodiments, a variant polypeptide includes anamino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity or similarity to a corresponding sequence of areference polypeptide. In certain embodiments, the amino acid additionsor deletions occur at the C-terminal end and/or the N-terminal end ofthe reference polypeptide. In certain embodiments, the amino aciddeletions include C-terminal truncations of about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about15, about 20, about 25, about 30, about 35, about 40, about 45, 50,about 55, about 60, about 65, about 70, about 75, about 80, about 85,about 90, about 95, about 100, about 105, about 110, about 115, about120, about 125, about 130, about 135, about 140, about 145, about 150,about 155, about 160, about 165, about 170, or about 175 or more aminoacids, including all intervening numbers of amino acids, e.g., 25, 26,27, 29, 30 . . . 100, 101, 102, 103, 104, 105 . . . 170, 171, 172, 173,174, etc.

As noted above, polypeptides of the presently disclosed subject mattermay be altered in various ways including amino acid substitutions,deletions, truncations, and insertions. Methods for such manipulationsare generally known in the art. For example, amino acid sequencevariants of a reference polypeptide can be prepared by mutations in theDNA. Methods for mutagenesis and nucleotide sequence alterations arewell known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad.Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Pat. No. 4,873,192, Watson, J. D. et al., MolecularBiology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park,Calif., 1987) and the references cited therein. Guidance as toappropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.).

As used herein, the term “substantially identical” refers to apolypeptide or a polynucleotide exhibiting at least 50% identity to areference amino acid sequence (for example, any one of the amino acidsequences described herein) or a nucleic acid sequence (for example, anyone of the nucleic acid sequences described herein). Preferably, such asequence is at least 60%, more preferably 80% or 85%, and morepreferably 90%, 95% or even 99% identical at the amino acid level ornucleic acid to the sequence used for comparison.

Sequence identity or homology is typically measured using sequenceanalysis software (for example, Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT,GAP, or PILEUP/PRETTYBOX programs). Such software matches identical orsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and/or other modifications. In an exemplaryapproach to determining the degree of identity or homology, a BLASTprogram may be used, with a probability score between e-3 and e-100indicating a closely related sequence. The percentage of identitybetween two sequences can also be determined with programs such asDNAMAN (Lynnon Biosoft, version 3.2). Using this program two sequencescan be. aligned using the optimal alignment algorithm (Smith andWaterman, 1981). After alignment of the two sequences the percentageidentity can be calculated by dividing the number of identicalnucleotides between the two sequences by the length of the alignedsequences minus the length of all gaps.

Terms that describe the orientation of polynucleotides include: 5′(normally the end of the polynucleotide having a free phosphate group)and 3′ (normally the end of the polynucleotide having a free hydroxyl(OH) group). Polynucleotide sequences can be annotated in the 5′ to 3′orientation or the 3′ to 5′ orientation.

As used herein, a “single guide RNA” or a “synthetic guide RNA” refersto the polynucleotide sequence comprising the guide sequence, the tracrsequence and the tracr mate sequence. The term “guide sequence” refersto the about 20 bp sequence within the guide RNA that specifies thetarget site and may be used interchangeably with the terms “guide” or“spacer”. The term “tracr mate sequence” may also be usedinterchangeably with the term “direct repeat(s)”.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature.

As used herein, the term “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

As used herein, the term “treating” or “treatment” refers to clinicalintervention in an attempt to alter the disease course of the individualor cell being treated, and can be performed either for prophylaxis orduring the course of clinical pathology. Therapeutic effects oftreatment include, without limitation, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastases, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Bypreventing progression of a disease or disorder, a treatment can preventdeterioration due to a disorder in an affected or diagnosed subject or asubject suspected of having the disorder, but also a treatment mayprevent the onset of the disorder or a symptom of the disorder in asubject at risk for the disorder or suspected of having the disorder.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like (e.g., which is to be the recipient of aparticular treatment, or from whom cells are harvested).

As used herein, the term “isolated cell” refers to a cell that isseparated from the molecular and/or cellular components that naturallyaccompany the cell. As used herein, the term “isolated” refers tomaterial that is free, substantially free, or essentially free tovarying degrees from components which normally accompany it as found inits native state. “Isolate” denotes a degree of separation from originalsource or surroundings.

As used herein, the term “cell population” refers to a group of at leasttwo cells expressing similar or different phenotypes. In non-limitingexamples, a cell population can include at least about 10, at leastabout 100, at least about 200, at least about 300, at least about 400,at least about 500, at least about 600, at least about 700, at leastabout 800, at least about 900, at least about 10³ cells, at least about10⁴ cells, at least about 10⁵ cells, at least about 10⁶ cells, at leastabout 10⁷ cells, or at least about 10⁸ cells expressing similar ordifferent phenotypes.

As used herein, the term “cleavage” refers to the breakage of thecovalent backbone of a DNA molecule. Cleavage can be initiated by avariety of methods including, but not limited to, enzymatic or chemicalhydrolysis of a phosphodiester bond. Both single-stranded cleavage anddouble-stranded cleavage are possible, and double-stranded cleavage canoccur as a result of two distinct single-stranded cleavage events. DNAcleavage can result in the production of either blunt ends or staggeredends. In certain embodiments, fusion polypeptides are used for targeteddouble-stranded DNA cleavage.

As used herein, the term “cleavage half-domain” refers to a polypeptidesequence which, in conjunction with a second polypeptide (eitheridentical or different) forms a complex having cleavage activity(preferably double-strand cleavage activity). The terms “first andsecond cleavage half-domains;” “+ and − cleavage half-domains” and“right and left cleavage half-domains” are used interchangeably to referto pairs of cleavage half-domains that dimerize.

As used herein, the term “chromosome” refers to a chromatin complexcomprising all or a portion of the genome of a cell. The genome of acell is often characterized by its karyotype, which is the collection ofall the chromosomes that comprise the genome of the cell. The genome ofa cell can comprise one or more chromosomes.

As used herein, the term “gene” includes a DNA region encoding a geneproduct, as well as all DNA regions which regulate the production of thegene product, whether or not such regulatory sequences are adjacent tocoding and/or transcribed sequences. Accordingly, a gene includes, butis not limited to, promoter sequences, terminators, translationalregulatory sequences such as ribosome binding sites and internalribosome entry sites, enhancers, silencers, insulators, boundaryelements, replication origins, matrix attachment sites and locus controlregions

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

A “functional region” or “functional portion” of a protein, polypeptideor nucleic acid is a protein, polypeptide or nucleic acid whose sequenceis not identical to the full-length protein, polypeptide or nucleicacid, yet retains the same function as the full-length protein,polypeptide or nucleic acid. A functional region can possess more,fewer, or the same number of residues as the corresponding nativemolecule, and/or can contain one or more amino acid or nucleotidesubstitutions. Methods for determining the function of a nucleic acid(e.g., coding function, ability to hybridize to another nucleic acid)are well-known in the art. Similarly, methods for determining proteinfunction are well-known. For example, the DNA-binding function of apolypeptide can be determined, for example, by filter-binding,electrophoretic mobility-shift, or immunoprecipitation assays. DNAcleavage can be assayed by gel electrophoresis. The ability of a proteinto interact with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical.

As used herein, the term “promoter” refers to a recognition site of apolynucleotide (DNA or RNA) to which an RNA polymerase binds. The term“enhancer” refers to a segment of DNA which contains sequences capableof providing enhanced transcription and in some instances can functionindependent of their orientation relative to another control sequence.An enhancer can function cooperatively or additively with promotersand/or other enhancer elements.

As used herein, the term “vector” refers to any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.,which is capable of replication when associated with the proper controlelements and which can transfer gene sequences into cells. Thus, theterm includes cloning and expression vehicles, as well as viral vectorsand plasmid vectors.

As used herein, the term “modulate” refers to altering positively ornegatively. Exemplary modulations include an about 1%, about 2%, about5%, about 10%, about 25%, about 50%, about 75%, or about 100% change.

As used herein, the term “increase” refers to alter positively by atleast about 5%, including, but not limited to, alter positively by about5%, by about 10%, by about 25%, by about 30%, by about 50%, by about75%, or by about 100%.

As used herein, the term “reduce” refers to alter negatively by at leastabout 5% including, but not limited to, alter negatively by about 5%, byabout 10%, by about 25%, by about 30%, by about 50%, by about 75%, or byabout 100%.

As used herein, the term “about” or “approximately” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 3 or more than 3 standarddeviations, per the practice in the art. Alternatively, “about” can meana range of up to 20%, preferably up to 10%, more preferably up to 5%,and more preferably still up to 1% of a given value. Alternatively,particularly with respect to biological systems or processes, the termcan mean within an order of magnitude, preferably within 5-fold, andmore preferably within 2-fold, of a value.

II. INSULATORS

Several cases of vector-related malignant transformation have beenreported in clinical settings, associated with the activation ofcellular oncogenes by vector-encoded enhancers (Baum et al. (2006),Nienhuis et al. (2006), Ramezani et al. (2006)) and various vectormodifications have been performed or proposed to reduce vectorgenotoxicity (Baum et al. (2006), Nienhuis et al. (2006), Ramezani etal. (2006)). A class of DNA elements known as chromatin insulators hasbeen recognized as one approach to improve vector safety and performance(Emery (2011)).

Insulators are naturally occurring DNA elements that help from thefunctional boundaries between adjacent chromatin domains. Insulatorsbind proteins that modify chromatin and alter regional gene expression.The placement of insulators in the vectors described herein offervarious potential benefits including, but not limited to, 1) shieldingof the vector from positional effect variegation of expression byflanking chromosomes (i.e., barrier activity, which may decreaseposition effects and vector silencing); and 2) shielding flankingchromosomes from insertional trans-activation of endogenous geneexpression by the vector (enhancer blocking). There are two basicclasses of chromatin insulators: (a) barrier insulators that block theencroachment of silencing heterochromatin into adjoining regions of openchromatin that are transcriptionally permissive, and (b) enhancerblocking insulators that prevent enhancer-mediated transcriptionalactivation of adjoining regions. The sequences that mediate theseactivities are physically separable and mechanistically distinct(Recillas-Targa et al. (2002)). Chromatin insulators do not exhibitinherent transcriptional enhancing or repressing activities on theirown. As such, they make ideal elements for reducing the interactionbetween gene transfer vectors and the target cell genome. Insulators canhelp to preserve the independent function of genes or transcriptionunits embedded in a genome or genetic context in which their expressionmay otherwise be influenced by regulatory signals within the genome orgenetic context (see, e.g., Burgess-Beusse et al. (2002) Proc. Nat'lAcad. Sci. USA, 99: 16433; and Zhan et al. (2001) Hum. Genet., 109:471).

The problems created by insertional mutagenesis of viral vectors arewidely known (Nienhuis (2013), Baum et al. (2006), Nienhuis et al.(2006)) as is the evidence that the risks of genotoxicity can be reducedby the use of chromatin insulators (Arumugam et al. (2007), Emery(2011), Evans-Galea et al. (2007), Rivella et al. (2000), Emery et al.(2000), Emery et al. (2002), Yannaki et al. (2002), Hino et al. (2004),Ramezani et al. (2003), Ramezani et al. (2008)). The presently disclosedsubject matter provides novel insulators that are powerful enhancerblocking insulators, and certain insulators additionally possess barrierinsulator activity. In vertebrates, the function of enhancer blockinginsulators is mediated through the zinc-finger DNA-binding factor CTCF(Gaszner and Felsenfeld (2006), Wallace and Felsenfeld (2007)). Ingeneral, these elements are thought to function through physical loopstructures, which are established by CTCF-mediated interactions betweenadjacent insulator elements or through CTCF-mediated tethering of thechromatin fiber to structural elements within the nucleus. The firstcharacterized vertebrate chromatin insulator is located within thechicken β-globin locus control region. This element, which contains aDNase-I hypersensitive site-4 (cHS4), appears to constitute the 5′boundary of the chicken β-globin locus (Prioleau et al. (1999) EMBO J.18: 4035-4048). A 1.2-kb region containing the cHS4 element displaysclassic insulator activities, including the ability to block theinteraction of globin gene promoters and enhancers in cell lines (Chunget al. (1993) Cell, 74: 505-514), and the ability to protect expressioncassettes in Drosophila (Id.), transformed cell lines (Pikaart et al.(1998) Genes Dev. 12: 2852-2862), and transgenic mammals (Wang et al.(1997) Nat. Biotechnol., 15: 239-243; Taboit-Dameron et al. (1999)Transgenic Res., 8: 223-235) from position effects. Much of thisactivity is contained in a 250-bp region. Within this stretch is a 49-bpcHS4 element (Chung et al. (1997) Proc. Natl. Acad. Sci., USA, 94:575-580) that interacts with the zinc finger DNA binding protein CTCFimplicated in enhancer-blocking assays (Bell et al. (1999) Cell, 98:387-396).

Insulators, such as cHS4, can block the interaction between enhancersand promoters when placed between these elements (Evans-Galea et al.(2007), Chung et al. (1997), Bell et al. (1999), Ryu et al. (2007), Ryuet al. (2008)). Several studies have demonstrated the ability of thecHS4 insulator to reduce position-effect silencing of gammaretroviralvectors (Evans-Galea et al. (2007), Rivella et al. (2000), Emery et al.(2000), Emery et al. (2002), Yannaki et al. (2002), Hino et al. (2004),Ramezani et al. (2006), Yao et al. (2003), Nishino et al. (2006), Akeret al. (2007), Li and Emery (2008)), and lentiviral vectors (Bank et al.(2005), Arumugam et al. (2007), Puthenveetil et al. (2004), Evans-Galeaet al. (2007), Ramezani et al. (2003), Aker et al. (2007), Ma et al.(2003), Chang et al. (2005), Pluta et al. (2005)). Those appropriatelydesigned studies demonstrated that inclusion of the 1.2 kb version ofthe cHS4 insulator increased the likelihood and/or consistency of vectortransgene expression in at least some settings (Arumugam et al. (2007),Emery (2011), Evans-Galea et al. (2007), Emery et al. (2002), Yannaki etal. (2002), Hino et al. (2004), Ramezani et al. (2006), Aker et al.(2007), Li and Emery (2008), Pluta et al. (2005). Jakobsson et al.(2004)). Nevertheless, the degree of protection afforded by the cHS4insulator is far from complete. In addition, the inclusion of the 1.2 KbcHS4 can adversely affect vector titers while the smallest cHS4 core hasbeen proven ineffective (Aker et al. (2007), Jakobsson et al. (2004)).By contrast, the insulators of the presently disclosed subject matter donot affect adversely the titers of viral vectors, and are more powerfuland effective than the cHS4 insulator.

The presently disclosed insulators are identified through genomicapproaches, e.g., using genomic approaches to identify insulators thatare powerful enhancer blockers as well as barrier insulators of thehuman genome. The presently disclosed insulators enhance the safety ofgene therapy (e.g., stem cell gene therapy, globin gene therapy). Forgene therapy of the hemoglobinopathies, powerful enhancers are requiredto achieve therapeutic levels of globin gene expression. Powerfulinsulators therefore represent one means to protect the genomicenvironment from the powerful enhancers of the integrating vectors.

The presently disclosed insulators possess powerful enhancer blockingactivity. For example, and not by way of limitation, an insulator of thepresent disclosure can reduce the activity of an enhancer element by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98% or atleast about 99%. In certain embodiments, the insulators possess barrieractivity in addition to enhancer blocking activity. The presentlydisclosed insulators substantially decrease the risks of insertionalmutagenesis and genotoxicity associated with viral vectors. Furthermore,when a presently disclosed insulator is incorporated into a vector, theinsulator does not adversely effect vector titers of the vector. Incertain embodiments, the insulators (e.g., insulator A1) increase the invivo expression of the globin gene or functional portion thereof.

In certain embodiments, the insulator comprises a Transcriptionalrepressor CTCF binding site, which has the nucleotide sequence set forthin SEQ ID NO: 18, which is provided below:

[SEQ ID NO: 18] CACCAGGTGGCGCT.

In one non-limiting embodiment, the insulator has the nucleotidesequence set forth in SEQ ID NO:1, which is provided below, or asequence which is at least about 95 percent homologous, or at leastabout 98 percent identical (homologous), to SEQ ID NO:1. This insulatorhaving the nucleotide sequence set forth in SEQ ID NO:1 is designated asinsulator A1.

[SEQ ID NO: 1] TCCTTCCTTTCTAAATGACGAGAGAGACAGAAGAATTCTTCAAGGTTAGTGTGTCCAGCATGCAACCTTTCCTTCCTGGATGAGCATCCCTGGAGTAGGAGAGCCAGCCTGCCTCCTGCGCTGGCACAGAGCCCGGTTCCCTAGACAACTGCCTCTCCAAATCTGATGTCCAGCGCCACCTGGTGTCCACATCAAGCAGACACAATTAATAGTCAACCTGTTCAGGAAAACTGTGAGGGGGAAAAAAAAGAAAGAGGATTTATGAAGGGAAAAGAAAGTTTAGAGGATATGCCACGATTG GCTAG

In certain embodiments, the insulator comprises a nucleotide sequence asset forth in SEQ ID NO:24, or a sequence which is at least about 95percent identical, or at least about 98 percent identical, to SEQ ID NO:24.

[SEQ ID NO: 24] CCAATC GTGGCATATC CTCTAAACTT TCTTTTCCCT TCATAAATCCTCTTTCTTTT TTTTCCCCCT CACAGTTTTC CTGAACAGGT TGACTATTAA TTGTGTCTGCTTGATGTGGA CACCAGGTGG CGCTGGACAT CAGATTTGGA GAGGCAGTTG TCTAGGGAACCGGGCTCTGT GCCAGCGCAG GAGGCAGGCT GGCTCTCCTA TTCCAGGGAT GCTCATCCAGGAAGGAAAGG TTGCATGCTG GACACACTAA CCTTGAAGAA TTCTTCTGTC TCTCTCGTCATTTAGAAAGG AAGGA.

In certain embodiments, the insulator comprises a nucleotide sequence asset forth in SEQ ID NO:25 (which is the reverse complement of SEQ IDNO:1), or a sequence which is at least about 95 percent identical, or atleast about 98 percent identical, to SEQ ID NO: 25.

[SEQ ID NO: 25] CTAGCCAATCGTGGCATATCCTCTAAACTTTCTTTTCCCTTCATAAATCCTCTTTCTTTTTTTTCCCCCTCACAGTTTTCCTGAACAGGTTGACTATTAATTGTGTCTGCTTGATGTGGACACCAGGTGGCGCTGGACATCAGATTTGGAGAGGCAGTTGTCTAGGGAACCGGGCTCTGTGCCAGCGCAGGAGGCAGGCTGGCTCTCCTACTCCAGGGATGCTCATCCAGGAAGGAAAGGTTGCATGCTGGACACACTAACCTTGAAGAATTCTTCTGTCTCTCTCGTCATTTAGAAAGG AAGGA

In certain embodiments, the insulator comprises a nucleotide sequence asset forth in hg18 coordinates 76229933 to 76230115 of chromosome 1.

In certain embodiments, the insulator comprises a nucleotide sequencebetween residues 68041 and 68160, or between residues and 68041 and68210, or between residues 68041 and 68280, or between residues 68005and 68305, of Homo sapiens chromosome 1 clone RP11-550H2, GenBankAccession No. AC092813.2, or a sequence at least 95 or 98 percentidentical thereto.

III. EXPRESSION CASSETTES

The presently disclosed subject matter provides expression cassettescomprising one or more the above-disclosed insulators (e.g., insulatorA1). In certain embodiments, an expression cassette comprises at leastone insulator having the nucleotide sequence set forth in SEQ ID NO:1,and a globin gene or a functional portion thereof operably linked to aβ-globin LCR region.

β-Globin LCR Region

The human β-globin gene cluster consists of five genes embedded withinone of many olfactory receptor gene arrays (Bulger et al., PNAS (1999);96:5129-5134). The cluster spans over 80 kb on chromosome 11p15.4, andincludes the five expressed β-like genes and cis-acting regulatoryelements that direct their stage-specific expression during ontogeny(Forget (2001), Molecular Mechanism of Beta Thalassemia. Steinberg M Het al., Eds. Disorders of Hemoglobin. Genetics, Pathophysiology andClinical Management, Cambridge University Press, Cambridge). The genesare arranged in the order of their developmental expression(Stamatoyannopoulos et al., (2001) Hemoglobin Switching. In:Stamatoyannopoulos G, et al., Eds. Molecular Basis of Blood Disorders,W.B. Saunders, Philadelphia, Pa.), 5′-ε-^(G)γ-^(A)γ-ψη-δ-β-3′. Theα-like globin gene cluster (5′-ξ2-ψξ 1-ψα2-ψα1-α2-α1-θ-3′) is locatedvery close to the telomere of the short arm of chromosome 16 and spansabout 40 kb. The expression of genes encoded within these twoindependent clusters is limited to erythroid cells and balanced so thatthe output of the β-globin-like chains matches that of the α-chains.This fine tuned balance is regulated at the transcriptional,posttranscriptional and posttranslational levels.

Developmental stage-specific expression is controlled by a number ofproximal or distal cis-acting elements and the transcriptional factorsthat bind to them. In the case of the β-globin gene (HBB), the proximalregulatory elements comprise the β-globin promoter and two downstreamenhancers, one located in the second intron of β-globin and the otherapproximately 800 bp downstream of the gene (Antoniou et al., EMBO J.(1988); 7:377-384; Trudel et al., Genes Dev. (1987); 1:954-961; Trudelet al., Mol. Cell. Biol. (1987); 7:4024-4029). The most prominent distalregulatory element is the β-globin LCR, located 50-60 kb upstream of theHBB and composed of several sub-regions with heightened sensitivity toDNaseI in erythroid cells (Forget (2001); Grosveld et al., Cell (1987);51:975-985; Talbot et al., Nature (1989); 338:352). The most prominentproperty of the LCR is its strong, transcription-enhancing activity. Anexemplary nucleotide sequence of the human β-globin region on chromosome11 is set forth in SEQ ID NO:19 (GenBank Access No.: NG_000007.31 whichis provided below:

[SEQ ID NO: 19]ggatcctcacatgagttcagtatataattgtaacagaataaaaaatcaattatgtattcaagttgctagtgtcttaagaggttcacatttttatctaactgattatcacaaaaatacttcgagttacttttcattataattcctgactacacatgaagagactgacacgtaggtgccttacttaggtaggttaagtaatttatccaaaaccacacaatgtagaacctaagctgattcggccatagaaacacaatatgtggtataaatgagacagagggatttctctccttcctatgctgtcagatgaatactgagatagaatatttagttcatctatcacacattaaacgggactttacatttctgtctgttgaagatttgggtgtggggataactcaaggtatcatatccaagggatggatgaaggcaggtgactctaacagaaagggaaaggatgttggcaaggctatgttcatgaaagtatatgtaaaatccacattaagcttctttctgcatgcattggcaatgtttatgaataatgtgtatgtaaaagtgtgctgtatattcaaaagtgtttcatgtgcctaggggtgtcaaatactttgagtttgtaagtatatacttctctgtaatgtgtctgaatatctctatttacttgattctcaataagtaggtatcatagtgaacatctgacaaatgtttgaggaacaatttagtgtttacctattcaccaaaatttattaaatgcctaatctgtatcagatatacaattatctggcgaaatctgtaattcctaatttaaacagctgtgtagcctaattagggataaaggcatgcaaacccataatttgtgtaggttgaaatgagctatagaaaaatgcagtatatttatcagaagtctttagggtcatgaaaaggaatggtcaactgacactgccagggactcatatgtaagagataactaatgtgaagtgactttaaaggagaaattagcagaagttttctttccatgtctcctcatcatgttacaataacggaagagattaaaacaacaaatacatttagacagcaatgtttatcctggttagatgttttaatctaaatctatcttggagtgttaaaatgcatttgctcacctactttaaaatataaatgaaggtaggaacctgtagatacaaaaagttggagaaaaaaagacaataaagatgacaaaaatctattaatccttgatagaaaatgagaagagataaaacactggtttacataaagaaaataagatggatagatagcagatccttataaaagtgataatttgagaaaaaaaatactccatattctgagtttcttcacataaaataatacaaatctgctgtggtaagttacaaagagatagattttttatcattatataaaagatattttaaacagagttatacaacaaaggaacagactatgtcatatattctcacttatcactataaacatctcagaaaaatctgcaaaatcatttcatagcattttaaatagttaggaataatgtagaaaactgaaacagttctaagtttcccacaaacttagagtctcaaatgttgcattacctaacttacctgcaaatattttatacaaatttgcacatgctactctagtcaaaaatatatgtacattatgggtattttctgtgtgtaacttggttctagttgcttctttcagaaatagcctctatttttgatttacctgataaaatcacattcctctccaaagccttctaaatacttccagactaactactttttagtacatctaagaagaaaagagttttgtctcttatccacctctgagtcaaaaagcagcatgtccatcaattggtacatagttcccacagccccacttagctctggattggagttctacttggcattgtttgcaactacatggacgtaaaatgcatggattctcttgaaaaaatgtttctgccatgatgttctctgaaagagactaaccttccctcgctttgcagagaaagactcgtgtaatccttgacaatgtcatctcatctatttattcccatgtctacccatatgtgaccttcatgtctttgctctaagcccctacatcctcaatctacacactaggatagtataaaagtaatagtaataatagtagtaatagtaataacaatacaatgattatggcttatactatacacaagacactgttgatatattatttcatttagtattcacagtaactctgtgcctcaagtactattgtaataccctttaagaggaggaaactgaggcacagggccctaaagtaatattccaagatgaagtggctactaactgacagagggcataattcaactcatgatatttggctctagaatacatgctctgaatcattatacaataataattcatgaggaaacattttttaaagcctaagttatttgctctgaaataagacataatttggggtgagaaagcttagattccatgaagtattacagcatttggtagtctttttgcactccaggtcttatttttactgcttaaacataataaaacatatggttcagtatgcctttgattttacaataatattcctgttatttttggaagcacagggtgtgggataatgctaattactagtgattagtattgagaggtgacagcgtgctggcagtcctcacagccctcgctcgctcttggcgcctcctctgcctgggctcccacattggtggcacttgaggagcccttcagccggccgctgcactgtgggagcccttttctgggctggccaaggccagagccggctccctcagcttgccaggaggtgtggagggacagacgcgggcaggaaccgggctgtgcgccgtgcttgagggagttccgggtgggcatgggctccgaggaccccgcactcggagccgccagccggccccaccggccgcgggcagtgaggggcttagcacctgggccagcagctgctgtgctcaattcctcgccgggccttagctgccttcctgcggggcagggctcgggacctgcagcgcgccatgcctgagcctccccaccttcatgggctcctgtgcggcccgagcctcgccgacgagcgccgccccctgctccagggcacccagtcccatcgaccacccaagggctgaagagtgcgggcgcacggcaggggactggcaggcagctccccctgcagcccaggtgcgggatccactgggtgaagccggctaggctcctgagtttgctggggatgcgaagaacccttatgtctagataagggattgtaaatacaccaattggcactctgtatctagctcaaggtttgtaaacacaccaatcagcaccctgtgtctagctcagggtttgtgaatgcaccaatcaacactctatctagctactctggtggggccttggagaacctttatgtctagctcagggattgtaaatacaccaatcggcagtctgtatctagctcaaggtttgtaaacacaccaatcagcaccctgtgtctagctcagggtttgtgaatgcaccaatcaacactctgtatctagctactctggtggggacgtggagaacctttatgtctagctcagggattgtaaatacaccactcggcagtctgtatctagctcaaggtttgtaaacacaccaatcagcaccctgtgtctagctcagggtttgtgaatgcaccaatcaacactctgtatctagctactctggtggggacttggagaacctttgtgtggacactctgtatctagctaatctggtggggacgtggagaacctttgtgtctagctcatggattgtaaatgcaccaatcagtgccctgtcaaaacagaccactgggctctaccaatcagcaggatgtgggtggggccagataagagaataaaagcaggctgcccgagccagcagtggcaacccgctcgggtccccttccacactgtggaagctttgttctttcgctctttgcaataaatcttgctgctgctcactgtttgggtctacactgcctttatgagctgtaacgctcaccgcgaaggtctgcagcttcactcttgaagccagcgagaccacgaacccaccgggaggaacgaacaactccagaggcgccgccttaagagctggaacgttcactgtgaaggtctgcagcttcactcctgagccagcgagaccacgaacccatcagaaggaagaaactccgaacacatccaaacatcagaacgaacaaactccacacacgcagcctttaagaactgtaacactcaccacgagggtccccggcttcattcttgaagtcagtgaaaccaagaacccaccaattccggacacagtatgtcagaaacaatatgagtcactaaatcaatatacttctcaacaatttccaacagcccttgcaattaacttggccatgtgactggttgtgactaaaataatgtggagataataatgtgttactccctaaggcagagtgcccttctatcattctctttcccttcctctatgtggcagaaagtaaaagattctgaaatgataaagtcaatcacaggaaggcacctggactcctggcccactgcttggaggagagcactcaggaccatgaacatctgactgtgacgtagcaataaagaaacccacgtttcatatgaaactgcttaaaattaatggcacaagtcatgtttttgatgttgcacatttgtctttatttgtggcttgttttgcttccacatcaatccactcaaggcctacattctgctataatgcaatttcaagttctttacaggccgagaaaaatgaatctgaattcctgacctccaaaagtgatcaagatatttttagttcaggctccaaaattttctcattttcataggttttcctcgattgatcattattcatgatttgcaaggaatcattcaatgttttctaaatctattactgcatcctgacacatatgacattttaactatgttccag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Five 5′ hypersensitive site (HS) sites (HS1-HS5) and one 3′ HS site havebeen identified in the human β-globin LCR (Stamatoyannopoulos et al.,(2001)). The 5′ HSs 1-4 are Dnase I hypersensitive sites. The HS2 andHS3 elements are the most powerful single elements within the LCR (Elliset al., EMBO J. (1996), 15:562-568; Collis et al., EMBO J. (1990)9:233-240), as corroborated by many groups. Deleting HS2 in the contextof βYAC in transgenic mice severely affects HS site formation as well asexpression of all of the human β-globin genes at every developmentalstage (Bungert et al., Mol. Cell Biol. (1999); 19:3062-3072). It wasreported that deletion of HS2 minimally reduced the expression of theembryonic εy and Phi globin genes in yolk sac-derived erythrocytes (Leyet al., Ann. N.Y. Acad. Sci. (1998); 850:45-53; Hug et al., Mol. CellBiol. (1996); 26:2906-2912). HS2 functions primarily as an enhancer.

In certain embodiments, the β-globin LCR region comprises a HS2 region.In non-limiting example, the β-globin LCR region comprises a HS2 region,a HS3 region, and a HS4 region. In certain embodiments, the HS2 region,HS3 region and HS4 region within the β-globin LCR region are contiguous.In one non-limiting embodiment, the β-globin LCR region consistingessentially of a HS2 region, a HS3 region and a HS4 region. In anotherembodiment, the β-globin LCR region comprises two introduced GATA-1binding sites at the junction between the HS3 region and the HS4 region.The HS3 region can lie between the HS2 region and the HS4 region. Thelength and the sequence of the HS2 region can vary. The HS2 region canhave a length of from about 400 bp to about 1000 bp, e.g., from about400 bp to about 500 bp, from about 500 bp to about 600 bp, from about600 bp to about 700 bp, from about 700 bp to about 800 bp, from about800 bp to about 900 bp, or from about 900 bp to about 1000 bp. In onenon-limiting embodiment, the HS2 region has a length of 860 bp. In onenon-limiting example, the HS2 region has the nucleotide sequence setforth in SEQ ID NO:9, which is provided below:

[SEQ ID NO: 9] GTATATGTGTATATATATATATATATATTCAGGAAATAATATATTCTAGAATATGTCACATTCTGTCTCAGGCATCCATTTTCTTTATGATGCCGTTTGAGGTGGAGTTTTAGTCAGGTGGTCAGCTTCTCCttttttttGCCATCTGCCCTGTAAGCATCCTGCTGGGGACCCAGATAGGAGTCATCACTCTAGGCTGAGAACATCTGGGCACACACCCTAAGCCTCAGCATGACTCATCATGACTCAGCATTGCTGTGCTTGAGCCAGAAGGTTTGCTTAGAAGGTTACACAGAACCAGAAGGCGGGGGTGGGGCACTGACCCCGACAGGGGCCTGGCCAGAACTGCTCATGCTTGGACTATGGGAGGTCACTAATGGAGACACACAGAAATGTAACAGGAACTAAGGAAAAACTGAAGCTTATTTAATCAGAGATGAGATGCTGGAAGGGATAGAGGGAGCTGAGCTTGTAAAAAGTATAGTAATCATTCAGCAAATGGTTTTGAAGCACCTGCTGGATGCTAAACACTATTTTCAGTGCTTGAATCATAAATAAGAATAAAACATGTATCTTATTCCCCACAAGAGTCCAAGTAAAAAATAACAGTTAATTATAATGTGCTCTGTCCCCCAGGCTGGAGTGCAGTGGCACGATCTCAGCTCACTGCAACCTCCGCCTCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCACCCTAATAGCTGGGATTACAGGTGCACACCACCATGCCAGGCTAATTTTTGTACTTTTTGTAGAGGCAGGGTATCACCATGTTGTCCAAGATGGTCTTGAACTCCTGAGCTCCAAGCAGTCCACCCACCTCAGCCTC CCAAAGTGCT

In certain embodiments, the HS2 region has a length of about 840 bp. Incertain embodiments, the HS2 region has a length of about 650 bp (e.g.,646 bp). In certain embodiments, the HS2 region has a length of about420 bp (e.g., 423 bp).

The length and the sequence of the HS3 region can vary. The HS3 regioncan have a length of from about 200 bp to about 1400 bp, e.g., fromabout 200 bp to about 300 bp, from about 300 bp to about 400 bp, fromabout 400 bp to about 500 bp, from about 500 bp to about 600 bp, fromabout 600 bp to about 700 bp, from about 700 bp to about 800 bp, fromabout 800 bp to about 900 bp, from about 900 bp to about 1000 bp, fromabout 1000 bp to about 1100 bp, from about 1100 bp to about 1200 bp,from about 1200 bp to about 1300 bp, or from about 1300 bp to about 1400bp. In certain embodiments, the HS3 region has a length of about 1300bp. In one non-limiting embodiment, the HS3 region has a length of 1308bp. In one non-limiting embodiment, the HS3 region has a length of 1301bp. In one non-limiting example, the HS3 region has the nucleotidesequence set forth in SEQ ID NO:5, which is provided below:

[SEQ ID NO: 5] AAGCTTTCATTAAAAAAAGTCTAACCAGCTGCATTCGACTTTGACTGCAGCAGCTGGTTAGAAGGTTCTACTGGAGGAGGGTCCCAGCCCATTGCTAAATTAACATCAGGCTCTGAGACTGGCAGTATATCTCTAACAGTGGTTGATGCTATCTTCTGGAACTTGCCTGCTACATTGAGACCACTGACCCATACATAGGAAGCCCATAGCTCTGTCCTGAACTGTTAGGCCACTGGTCCAGAGAGTGTGCATCTCCTTTGATCCTCATAATAACCCTATGAGATAGACACAATTATTACTCTTACTTTATAGATGATGATCCTGAAAACATAGGAGTCAAGGCACTTGCCCCTAGCTGGGGGTATAGGGGAGCAGTCCCATGTAGTAGTAGAATGAAAAATGCTGCTATGCTGTGCCTCCCCCACCTTTCCCATGTCTGCCCTCTACTCATGGTCTATCTCTCCTGGCTCCTGGGAGTCATGGACTCCACCCAGCACCACCAACCTGACCTAACCACCTATCTGAGCCTGCCAGCCTATAACCCATCTGGGCCCTGATAGCTGGTGGCCAGCCCTGACCCCACCCCACCCTCCCTGGAACCTCTGATAGACACATCTGGCACACCAGCTCGCAAAGTCACCGTGAGGGTCTTGTGTTTGCTGAGTCAAAATTCCTTGAAATCCAAGTCCTTAGAGACTCCTGCTCCCAAATTTACAGTCATAGACTTCTTCATGGCTGTCTCCTTTATCCACAGAATGATTCCTTTGCTTCATTGCCCCATCCATCTGATCCTCCTCATCAGTGCAGCACAGGGCCCATGAGCAGTAGCTGCAGAGTCTCACATAGGTCTGGCACTGCCTCTGACATGTCCGACCTTAGGCAAATGCTTGACTCTTCTGAGCTCAGTCTTGTCATGGCAAAATAAAGATAATAATAGTGTTTTTTTATGGAGTTAGCGTGAGGATGGAAAACAATAGCAAAATTGATTAGACTATAAAAGGTCTCAACAAATAGTAGTAGATTTTATCATCCATTAATCCTTCCCTCTCCTCTCTTACTCATCCCATCACGTATGCCTCTTAATTTTCCCTTACCTATAATAAGAGTTATTCCTCTTATTATATTCTTCTTATAGTGATTCTGGATATTAAAGTGGGAATGAGGGGCAGGCCACTAACGAAGAAGATGTTTCTCAAAGAAGCCATTCTCCCCACATAGATCATCTCAGCAGGGTTCAGGAAGATAAAGGAGGATCAAGGTCGAAGGTAGGAACTAAGGAAGAACACTGGGCAAGTGGATC C

In certain embodiments, the HS3 region has a length of about 850 bp(e.g., 845 bp). In certain embodiments, the HS3 region has a length offrom about 280 bp to about 290 bp (e.g., 280 bp and 287 bp).

Similarly, the length and the sequence of the HS4 region can vary. TheHS4 region can have a length of from about 200 bp to about 1200 bp,e.g., from about 200 bp to about 300 bp, from about 300 bp to about 400bp, from about 400 bp to about 500 bp, from about 500 bp to about 600bp, from about 600 bp to about 700 bp, from about 700 bp to about 800bp, from about 800 bp to about 900 bp, from about 900 bp to about 1000bp, from about 1000 bp to about 1100 bp, or from about 1100 bp to about1200 bp.

In certain embodiments, the HS4 region has a length of about 1.0 kb ormore. In certain embodiments, the HS4 region has a length of about 1.1kb. In certain embodiments, the HS4 region has a length of about 1150 bp(e.g., 1153 bp). In one non-limiting embodiment, the HS4 region has alength of 1065 bp. In one non-limiting example, the HS4 region has thenucleotide sequence set forth in SEQ ID NO:6, which is provided below:

[SEQ ID NO: 6] TGAGCCCCTTTTCCTCTAACTGAAAGAAGGAAAAAAAAAATGGAACCCAAAATATTCTACATAGTTTCCATGTCACAGCCAGGGCTGGGCAGTCTCCTGTTATTTCTTTTAAAATAAATATATCATTTAAATGCATAAATAAGCAAACCCTGCTCGGGAATGGGAGGGAGAGTCTCTGGAGTCCACCCCTTCTCGGCCCTGGCTCTGCAGATAGTGCTATCAAAGCCCTGACAGAGCCCTGCCCATTGCTGGGCCTTGGAGTGAGTCAGCCTAGTAGAGAGGCAGGGCAAGCCATCTCATAGCTGCTGAGTGGGAGAGAGAAAAGGGCTCATTGTCTATAAACTCAGGTCATGGCTATTCTTATTCTCACACTAAGAAAAAGAATGAGATGTCTACATATACCCTGCGTCCCCTCTTGTGTACTGGGGCCCCCAAGAGCTCTCTAAAAGTGATGGCAAAGTCATTGCGCTAGATGCCATCCCATCTATTATAAACCTGCATTTGTCTCCACACACCAGTCATGGACAATAACCCTCCTCCCAGGTCCACGTGCTTGTCTTTGTATAATACTCAAGTAATTTCGGAAAATGTATTCTTTCAATCTTGTTCTGTTATTCCTGTTTCAATGGCTTAGTAGAAAAAGTACATACTTGTTTTCCCATAAATTGACAATAGACAATTTCACATCAATGTCTATATGGGTCGTTGTGTTTGCTGTGTTTGCAAAAACTCACAATAACTTTATATTGTTACTACTCTAAGAAAGTTACAACATGGTGAATACAAGAGAAAGCTATTACAAGTCCAGAAAATAAAAGTTATCATCTTGAGGCCTCAGCTTTCTAGGAATAATATCAATATTACAAAATTTAATCTAACAATTATGAACAGCAATGAGATAATATGTACAAAGTACCCAGACCTATGTGGTAGAGCATCAAGGAAGCGCATTGCGGAGCAGTTTTTTGTTTGTTTGTTTTTGTATTCTGTTTCGTGAGGCAAGGTTTCACTCTGCTGTCCAGGCTGGAGTGCAGTGGCAAGATCATGTCT CACTGCAGCCTTGAC

In one non-limiting example, the HS4 region has the nucleotide sequenceset forth in SEQ ID NO:7, which is provided below:

[SEQ ID NO: 7] TGAGCCCCTTTTCCTCTAACTGAAAGAAGGAAAAAAAAAATGGAACCCAAAATATTCTACATAGTTTCCATGTCACAGCCAGGGCTGGGCAGTCTCCTGTTATTTCTTTTAAAATAAATATATCATTAAATGCATAAATAAGCAAACCCTGCTCGGGAATGGGAGGGAGAGTCTCTGGAGTCCACCCCTTCTCGGCCCTGGCTCTGCAGATAGTGCTATCAAAGCCCTGACAGAGCCCTGCCCATTGCTGGGCCTTGGAGTGAGTCAGCCTAGTAGAGAGGCAGGGCAAGCCATCTCATAGCTGCTGAGTGGGAGAGAGAAAAGGGCTCATTGTCTATAAACTCAGGTCATGGCTATTCTTATTCTCACACTAAGAAAAAGAATGAGATGTCTACATATACCCTGCGTCCCCTCTTGTGTACTGGGGCCCCCAAGAGCTCTCTAAAAGTGATGGCAAAGTCATTGCGCTAGATGCCATCCCATCTATTATAAACCTGCATTTGTCTCCACACACCAGTCATGGACAATAACCCTCCTCCCAGGTCCACGTGCTTGTCTTTGTATAATACTCAAGTAATTTCGGAAAATGTATTCTTTCAATCTTGTTCTGTTATTCCTGTTTCAATGGCTTAGTAGAAAAAGTACATACTTGTTTTCCCATAAATTGACAATAGACAATTTCACATCAATGTCTATATGGGTCGTTGTGTTTGCTGTGTTTGCAAAAACTCACAATAACTTTATATTGTTACTACTCTAAGAAAGTTACAACATGGTGAATACAAGAGAAAGCTATTACAAGTCCAGAAAATAAAAGTTATCATCTTGAGGCCTCAGCTTTCTAGGaATAATATCAATATTACAAAATTAATCTAACAATTATGAACAGCAATGAGATAATATGTACAAAGTACCCAGACCTATGTGGTAGAGCATCAAGGAAGCGCATTGCGGAGCAGTTTTTTGTTTGTTTGTTTTTGTATTCTGTTTCGTGAGGCAAGGTTTCACTCTGCTGTCCAGGCTGGAGTGCAGTGGCAAGATCATGTCTCA CTGCAGCCTTGACAC

In certain embodiments, the HS4 region has a length of less than about1.0 kb, e.g., less than about 900 bp, less than about 700 bp, less thanabout 600 bp, or less than about 500 bp. In certain embodiments, the HS4region has a length of less than about 500 bp. In certain embodiments,the HS4 region has a length of about 450 bp. In one non-limitingembodiment, the HS4 region has a length of about 446 bp. In onenon-limiting example, the HS4 region has the nucleotide sequence setforth in SEQ ID NO:8, which is provided below:

[SEQ ID NO: 8] TGGAACCCAAAATATTCTACATAGTTTCCATGTCACAGCCAGGGCTGGGCAGTCTCCTGTTATTTCTTTTAAAATAAATATATCATTTAAATGCATAAATAAGCAAACCCTGCTCGGGAATGGGAGGGAGAGTCTCTGGAGTCCACCCCTTCTCGGCCCTGGCTCTGCAGATAGTGCTATCAAAGCCCTGACAGAGCCCTGCCCATTGCTGGGCCTTGGAGTGAGTCAGCCTAGTAGAGAGGCAGGGCAAGCCATCTCATAGCTGCTGAGTGGGAGAGAGAAAAGGGCTCATTGTCTATAAACTCAGGTCATGGCTATTCTTATTCTCACACTAAGAAAAAGAATGAGATGTCTACATATACCCTGCGTCCCCTCTTGTGTACTGGGGTCCCCAAGAGCTCTCTAAAAGTGATGGCAAAGTCATTGCGCTAGATGCCATCCCATCT

In certain embodiments, the HS4 region has a length of about 280 bp(e.g., 283 bp). In certain embodiments, the HS4 region has a length ofabout 240 bp (e.g., 243 bp).

In certain non-limiting embodiments, the β-globin LCR region comprises aHS2 region having the nucleotide sequence set forth in SEQ ID NO:9, SEQID NO:20 or SEQ ID NO:21, a HS3 region having the nucleotide sequenceset forth in SEQ ID NO:5, and a HS4 region having the nucleotidesequence set forth in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

In one non-limiting embodiment, the β-globin LCR region comprises a HS2region having the nucleotide sequence set forth in SEQ ID NO:9, a HS3region having the nucleotide sequence set forth in SEQ ID NO:5, and aHS4 region having the nucleotide sequence set forth in SEQ ID NO:7, asshown in FIG. 1.

In another non-limiting embodiment, the β-globin LCR region furthercomprises a HS1 region, i.e., a β-globin LCR region comprising a HS1region, a HS2 region, a HS3 region, and a HS4 region. In certainembodiments, the HS1 region, HS2 region, HS3 region and HS4 regionwithin the β-globin LCR region are contiguous. In one non-limitingembodiment, the β-globin LCR region consisting essentially of a HS1region, a HS2 region, a HS3 region and a HS4 region. In anotherembodiment, the β-globin LCR region comprises two introduced GATA-1binding sites at the junction between the HS3 region and the HS4 region.

The length and the sequence of the HS1 region can vary. In certainembodiments, the HS1 region is from about 300 bp to about 1500 bp inlength, e.g., from about 300 bp to about 1100 bp in length. In certainembodiments, the HS1 region has a length of about 1.0 kb or more, e.g.,about 1.1 kb, about 1.2 kb, about 1.3 kb, about 1.4 kb, or about 1.5 kb.In certain embodiments, the HS1 region has a length of about 1.1 kb. Inone non-limiting example, the HS1 region has a length of 1074 bp. In onenon-limiting example, the HS1 region has the nucleotide sequence setforth in SEQ ID NO:2, which is provided below:

[SEQ ID NO: 2] AAGTAAACTTCCACAACCGCAAGCTTATTGAGGCTAAGGCATCTGTGAAGGAAAGAAACATCTCCTCTAAACCACTATGCTGCTAGAGCCTCTTTTCTGTACTCAAGCCTCATTCAGACACTAGTGTCACCAGTCTCCTCATATACCTATTGTATTTTCTTCTTCTTGCTGGTTTAGTCATGTTTTCTGGGAGCTTAGGGGCTTATTTTATTTTGTTTTGTTTTCTAATCAACAGAGATGGGCAAACCCATTATTTTTTTCTTTAGACTTGGGATGGTGATAGCTGGGCAGCGTCAGAAACTGTGTGTGGATATAGATAAGAGCTCGGACTATGCTGAGCTGTGATGAGGGAGGGACCTAGCCAAAGGCAGTGAGAGTCAGAATGCTCCTGCTATTGCCTTCTCAGTCCCCACGCTTGGTTTCTACACAAGTAGATACATAGAAAAGGCTATAGGTTAGTGTTTGAGAGTCCTGCATGAGTTAGTTGCTCAGAAATGCCCGATAAATATGTTATGTGTGTTTATGTATATATATGTTTTATATATATATATGTGTGTGTGTGTGTGTGTGTGTGTTGTGTTTACAAATATGTGATTATCATCAAAACGTGAGGGCTAAAGTGACCAGATAACTTGCAGGTCCTAGGATACCAGGAAAATAAATTACATTCCAAAAATTTAACTGAGACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAACCAGTGATCCATGGACACAGGGAGGGGAACATCACACACTGGGGCCTGTTGGGGGTGGGGGGCTAGGGGAAGGATAGCATTAGGAGAAATACCTAATGTAGATGACGGGTTGATGGGTGCAGCAAACCACCATGGCACATGTACCCCAGAACTTAAAGCATATTAAAAAAACAGTGATCATAAAAGAAGCTCAAATTTAACTATAAGAGACGGAATGGCTCCCACAATTCTTAACTATAATCTTACAGAATATTCTCATTGAATAGAAGTATGCTTATCATTAGAGATTTGGACAGCCAGGAAAGCACAGAAAAAAAAAAAAGGAGCTCTGTTGCCTTATAGCCTAGAGGTGTTT

In certain embodiments, the HS1 region has a length of less than about1.0 kb, e.g., from about 400 bp to about 700 bp, from about 400 bp toabout 500 bp, from about 500 bp to about 600 bp, from about 600 bp toabout 700 bp, from about 700 bp to about 800 bp, from about 800 bp toabout 900 bp, or from about 900 bp to about 1.0 kb. In certainembodiments, the HS1 region has a length of less than about 700 bp. Incertain embodiments, the HS1 region has a length of about 600 bp. In onenon-limiting embodiment, the HS1 region has a length of 602 bp. In onenon-limiting example, the HS1 region has the nucleotide sequence setforth in SEQ ID NO:3, which is provided below:

[SEQ ID NO: 3] GGCATCTGTGAAGGAAAGAAACATCTCCTCTAAACCACTATGCTGCTAGAGCCTCTTTTCTGTACTCAAGCCTCATTCAGACACTAGTGTCACCAGTCTCCTCATATACCTATTGTATTTTCTTCTTCTTGCTGGTTTAGTCATGTTTTCTGGGAGCTTAGGGGCTTATTTTATTTTGTTTTGTTTTCTAATCAACAGAGATGGGCAAACCCATTATTTTTTTCTTTAGACTTGGGATGGTGATAGCTGGGCAGCGTCAGAAACTGTGTGTGGATATAGATAAGAGCTCGGACTATGCTGAGCTGTGATGAGGGAGGGACCTAGCCAAAGGCAGTGAGAGTCAGAATGCTCCTGCTATTGCCTTCTCAGTCCCCACGCTTGGTTTCTACACAAGTAGATACATAGAAAAGGCTATAGGTTAGTGTTTGAGAGTCCTGCATGAGTTAGTTGCTCAGAAATGCCCGATAAATATGTTATGTGTGTTTATGTATATATATGTTTTATATATATATATGTGTGTGTGTGTGTGTGTGTGTGTTGTGTTTACAAATATGTGATTATCATCAAAACGTGAGGGCTAAAGTGACCAGATAACTTGCA GG

In certain embodiments, the HS1 region has a length of less than about500 bp. In certain embodiments, the HS1 region has a length of about 490bp. In one non-limiting embodiment, the HS1 region has a length of 489bp. In one non-limiting example, the HS1 region has the nucleotidesequence set forth in SEQ ID NO:4, which is provided below:

[SEQ ID NO: 4] GGCATCTGTGAAGGAAAGAAACATCTCCTCTAAACCACTATGCTGCTAGAGCCTCTTTTCTGTACTCAAGCCTCATTCAGACACTAGTGTCACCAGTCTCCTCATATACCTATTGTATTTTCTTCTTCTTGCTGGTTTAGTCATGTTTTCTGGGAGCTTAGGGGCTTATTTTATTTTGTTTTGTTTTCTAATCAACAGAGATGGGCAAACCCATTATTTTTTTCTTTAGACTTGGGATGGTGATAGCTGGGCAGCGTCAGAAACTGTGTGTGGATATAGATAAGAGCTCGGACTATGCTGAGCTGTGATGAGGGAGGGACCTAGCCAAAGGCAGTGAGAGTCAGAATGCTCCTGCTATTGCCTTCTCAGTCCCCACGCTTGGTTTCTACACAAGTAGATACATAGAAAAGGCTATAGGTTAGTGTTTGAGAGTCCTGCATGAGTTAGTTGCTCAGAAATGCCCGATAAATATGTTATGTGTGTTTATGT

Recent studies have shown that HS2 is not erythroid-specific, but isexpressed in other cell lines and lineages (See Example 3 and FIG. 7)and is also present in undifferentiated human embryonic stem cells(Chang et al., Stem cell reviews (2013); 9:397-407). Due to thenon-erythroid activity of HS2, HS2-containing globin vectors may pose arisk for their safe use in clinical treatment, e.g., for treatingthalassemia and sickle cell patients. In certain embodiments, theβ-globin LCR region does not comprise a HS2 region. In certainembodiments, the β-globin LCR region does not comprise a core sequenceof HS2. A core sequence of HS2 provides position independent, high levelexpression. In addition, a core sequence of HS2 sustains the enhanceractivity of HS2. For example, the core sequence of HS2 enhances thetranscription of a globin gene (e.g., human β-globin gene).Additionally, a core sequence of HS2 comprises one or more binding sitesor binding motifs for ubiquitous as well as tissue-specific (e.g.,erythroid-specific) proteins (e.g., transcription factors), including,but not limited to, members of AP1 family of proteins (e.g., NF-E2),GATA-1 (also known as “NF-E1” or “NFE1”), Krüppel-like Zn fingerproteins (e.g., ubiquitous proteins Sp1 and YY1, anderythroid-restricted factor erythroid Krüppel-like factor (EKLF)), andbasic helix-loop-helix (bHLH) proteins (E boxes) (e.g., USF and TAL1).AP1 binding sites are required for enhancement and induction (Moi andKan (1990); Ney et al., (1990); Talbot and Grosveld (1991)).Furthermore, binding of NF-E2 can cause disruption of in vitroreconstituted chromatin at HS2 (Armstrong and Emerson (1996)). Mutationsin the GATA-1 binding sites can cause a reduction in enhancer activityof HS2 in transgenic mice (Caterina et al., (1994)). Although both AP1(e.g., AP1/NF-E2) and GATA1 binding sites are important for corefunction, mice lacking these factors do not show impaired globin geneexpression (Weiss et al., 1994).

In certain embodiments, the β-globin LCR region does not comprise thefull length of a core sequence of HS2. In certain embodiments, the coresequence of a HS2 region is a core sequence of human HS2. In onenon-limiting embodiment, the core sequence of human HS2 comprises atandem pair of binding sites for members of AP1 family of proteins(e.g., NF-E2) (referred to as “AP1/NF-E2” binding sites) (e.g.,GCTGAGTCA, and GATGAGTCA), one binding site for Kruppel-like Zn fingerproteins (e.g., AGGGTGTGT), one GATA-1 binding site (e.g., CTATCT), andthree E boxes (CANNTG, e.g., CAGATG, and CACCTG). In one non-limitingembodiment, the β-globin LCR region does not comprise the full length ofa 388 bp core sequence of human HS2, which has the nucleotide sequenceset forth in SEQ ID NO:20 provided below:

[SEQ ID NO: 20] TAAGCTTCAGTTTTTCCTTAGTTCCTGTTACATTTCTGTGTGTCTCCATTAGTGACCTCCCATAGTCCAAGCATGAGCAGTTCTGGCCAGGCCCCTGTCGGGGTCAGTGCCCCACCCCCGCCTTCTGGTTCTGTGTAACCTTCTAAGCAAACCTTCTGGCTCAAGCACAGCAATGCTGAGTCATGATGAGTCATGCTGAGGCTTAGGGTGTGTGCCCAGATGTTCTCAGCCTAGAGTGATGACTCCTATCTGGGTCCCCAGCAGGATGCTTACAGGGCAGATGGCAAAAAAAAGGAGAAGCTGACCACCTGACTAAAACTCCACCTCAAACGGCATCATAAAGAAAATGGATGCCTGAGACAGAATGTGACATATTCTAGAATATATT

The nucleotide sequence set forth in SEQ ID NO:20 corresponds tonucleotides position 16671 to position 17058 of SEQ ID NO:19 (GenBankAccess No.: NG 000007.3). In SEQ ID NO:20, one AP1/NF-E2 binding sitehaving the nucleotide sequence of GCTGAGTCA is located at position 175to position 183, one AP1/NF-E2 binding site having the nucleotidesequence of GATGAGTCA is located at position 185 to position 193, onebinding site for Kruppel-like Zn finger proteins having the nucleotidesequence of AGGGTGTGT is located as position 205 to position 213, two Eboxes, each of which have the nucleotide sequence of CAGATG, is locatedat position 217 to position 222, and position 278 to position 283, oneGATA-1 binding site having the nucleotide sequence of CTATCT is locatedat position 246 to position 251, one E box having the nucleotidesequence of CACCTG is located at position 306 to position 311.

In one non-limiting embodiment, the β-globin LCR region does notcomprise the full length of a 387 bp core sequence of human HS2, whichhas the nucleotide sequence set forth in SEQ ID NO:21 provided below:

[SEQ ID NO: 21] TAAGCTTCAGTTTTTCCTTAGTTCCTGTTACATTTCTGTGTGTCTCCATTAGTGACCTCCCATAGTCCAAGCATGAGCAGTTCTGGCCAGGCCCCTGTCGGGGTCAGTGCCCCACCCCCGCCTTCTGGTTCTGTGTAACCTTCTAAGCAAACCTTCTGGCTCAAGCACAGCAATGCTGAGTCATGATGAGTCATGCTGAGGCTAGGGTGTGTGCCCAGATGTTCTCAGCCTAGAGTGATGACTCCTATCTGGGTCCCCAGCAGGATGCTTACAGGGCAGATGGCAAAAAAAAGGAGAAGCTGACCACCTGACTAAAACTCCACCTCAAACGGCATCATAAAGAAAATGGATGCCTGAGACAGAATGTGACATATTCTAGAATATATT

In SEQ ID NO:21, one AP1/NF-E2 binding site having the nucleotidesequence of GCTGAGTCA is located at position 175 to position 183, oneAP1/NF-E2 binding site having the nucleotide sequence of GATGAGTCA islocated at position 185 to position 193, one binding site forKruppel-like Zn finger proteins having the nucleotide sequence ofAGGGTGTGT is located as position 204 to position 212, two E boxes, eachof which have the nucleotide sequence of CAGATG, is located at position216 to position 221, and position 277 to position 282, one GATA-1binding site having the nucleotide sequence of CTATCT is located atposition 245 to position 250, one E box having the nucleotide sequenceof CACCTG is located at position 305 to position 310.

In certain embodiments, the β-globin LCR region does not comprise a HS2region that comprises a core sequence of HS2. A HS2 region thatcomprises a core sequence of HS2 can vary in length and sequence. Innon-limiting examples, a HS2 region that comprises a core sequence ofHS2 is from about 400 bp to about 1000 bp, e.g., from about 400 bp toabout 500 bp, from about 500 bp to about 600 bp, from about 600 bp toabout 700 bp, from about 700 bp to about 800 bp, from about 800 bp toabout 900 bp, or from about 900 bp to about 1000 bp, in length. In onenon-limiting embodiment, the β-globin LCR region does not comprise a 840bp HS2 region (e.g., the HS2 region comprised in the globin vector TNS9disclosed in U.S. Pat. No. 7,541,179). In one non-limiting embodiment,the β-globin LCR region does not comprise a 860 bp HS2 region. In onenon-limiting embodiment, the β-globin LCR region does not comprise anabout 650 bp HS2 region. In one non-limiting example, the β-globin LCRregion does not comprise a 646 bp HS2 region (e.g., the HS2 regioncomprised in the globin vector LentiGlobin™, also known as “β⁸⁷”). Inone non-limiting embodiment, the β-globin LCR region does not comprisean about 420 bp HS2 region. In one non-limiting example, the β-globinLCR region does not comprise a 423 bp HS2 region (e.g., the HS2 regioncomprised in the globin vector disclosed in Sadelain et al., Proc. Nat'lAcad. Sci. (USA) (1995); 92:6728-6732).

In certain embodiments, the β-globin LCR region does not comprise a HS2region that sustains the enhancer activity of HS2. In certainembodiments, the β-globin LCR region does not comprise a HS2 region thatis capable of enhancing the transcription of a globin gene (e.g., humanβ-globin gene). In non-limiting examples, the β-globin LCR region doesnot comprise a HS2 region whose ability to enhance the transcription ofa globin gene (e.g., human β-globin gene) is no less than 60%, no lessthan 70%, no less than 80%, no less than 90%, or no less than 95% incomparison to a native HS2 region.

In certain embodiments, the β-globin LCR region does not comprise a HS2region that comprises one, two, three, four, five, six or seven of thefollowing binding sites: two (a tandem pair of) AP1/NF-E2 binding sites(e.g., GCTGAGTCA, and GATGAGTCA), one binding site for Kruppel-like Znfinger proteins (e.g., AGGGTGTGT), one GATA-1 binding site (e.g.,CTATCT), and three E boxes (CANNTG, e.g., CAGATG, and CACCTG). Incertain embodiments, the β-globin LCR region does not comprise a HS2region that comprises six of the above-described binding sites. Forexample, in certain embodiments, the β-globin LCR region does notcomprise a HS2 region that comprises two AP1/NF-E2 binding sites, onebinding site for Kruppel-like Zn finger proteins, one GATA-1 bindingsite, and two not three E boxes. In certain embodiments, the β-globinLCR region does not comprise a HS2 region that comprises one not twoAP1/NF-E2 binding site, one binding site for Kruppel-like Zn fingerproteins, one GATA-1 binding site, and three E boxes. In certainembodiments, the β-globin LCR region does not comprise a HS2 region thatcomprises two AP1/NF-E2 binding sites, one GATA-1 binding site, andthree E boxes and does not comprise one binding site for Kruppel-like Znfinger proteins. In certain embodiments, the β-globin LCR region doesnot comprise a HS2 region that comprises two AP1/NF-E2 binding sites,one binding site for Kruppel-like Zn finger proteins, and three E boxes,and does not comprise one GATA-1 binding site.

In certain embodiments, the β-globin LCR region comprises a HS1 region,a HS3 region, and a HS4 region, and does not comprise a HS2 region. Incertain embodiments, the HS1 region, HS3 region and HS4 region withinthe β-globin LCR region are contiguous. In one non-limiting embodiment,the β-globin LCR region consisting essentially of a HS1 region, a HS3region and a HS4 region. In another embodiment, the β-globin LCR regioncomprises two introduced GATA-1 binding sites at the junction betweenthe HS3 region and the HS4 region. The HS3 region can lie between theHS1 region and the HS4 region.

In certain non-limiting embodiments, the β-globin LCR region comprises aHS1 region having the nucleotide sequence set forth in SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:22 or SEQ ID NO:23, a HS3 region havingthe nucleotide sequence set forth in SEQ ID NO:5, and a HS4 regionhaving the nucleotide sequence set forth in SEQ ID NO:6, SEQ ID NO:7 orSEQ ID NO:8, and the β-globin LCR region does not comprise a HS2 region.

In one non-limiting embodiment, the β-globin LCR region comprises a HS1region having the nucleotide sequence set forth in SEQ ID NO:2, a HS3region having the nucleotide sequence set forth in SEQ ID NO:5, and aHS4 region having the nucleotide sequence set forth in SEQ ID NO:6, andthe β-globin LCR region does not comprise a HS2 region, as shown in FIG.2.

In one non-limiting embodiment, the β-globin LCR region comprises a HS1region having the nucleotide sequence set forth in SEQ ID NO:3, a HS3region having the nucleotide sequence set forth in SEQ ID NO:5, and aHS4 region having the nucleotide sequence set forth in SEQ ID NO:8, andthe β-globin LCR region does not comprise a HS2 region, as shown in FIG.3.

In one non-limiting embodiment, the β-globin LCR region comprises a HS1region having the nucleotide sequence set forth in SEQ ID NO:4, a HS3region having the nucleotide sequence set forth in SEQ ID NO:5, and aHS4 region having the nucleotide sequence set forth in SEQ ID NO:8, andthe β-globin LCR region does not comprise a HS2 region.

In certain embodiments, the β-globin LCR region does not comprise a HS1region or a HS2 region. In certain embodiments, the β-globin LCR regiondoes not comprise a core sequence of HS1. A core sequence of HS1sustains the activity of HS1, e.g., enhancer activity, or functioning asa facilitator or regulatory element to tether the enhancer activity ofother HS regions, e.g., HS2-4. In addition, a core sequence of HS1comprises one or more binding sites or binding motifs for ubiquitous aswell as tissue-specific (e.g., erythroid-specific) proteins (e.g.,transcription factors), including, but not limited to, GATA-1, andKruppel-like Zn finger proteins (e.g., erythroid-restricted factorEKLF).

In certain embodiments, the β-globin LCR region does not comprise thefull length of a core sequence of HS1. In certain embodiments, the coresequence of a HS1 region is a core sequence of human HS1. In onenon-limiting embodiment, the core sequence of human HS1 comprises twoGATA-1 binding sites (e.g., TTATCT, and CTATCA), and one binding sitefor EKLF (e.g., CCACACACA). In certain embodiments, the β-globin LCRregion does not comprise the full length of a 286 bp core sequence ofhuman HS1. In one non-limiting embodiment, the 286 bp core sequence ofhuman HS1 has the nucleotide sequence set forth in SEQ ID NO:22 providedbelow:

[SEQ ID NO: 22] CTGAGCAACTAACTCATGCAGGACTCTCAAACACTAACCTATAGCCTTTTCTATGTATCTACTTGTGTAGAAACCAAGCGTGGGGACTGAGAAGGCAATAGCAGGAGCATTCTGACTCTCACTGCCTTTGGCTAGGTCCCTCCCTCATCACAGCTCAGCATAGTCCGAGCTCTTATCTATATCCACACACAGTTTCTGACGCTGCCCAGCTATCACCATCCCAAGTCTAAAGAAAAAAATAATGGGTTTGCCCATCTCTGTTGATTAGAAAACAAAACAAAATAAAIn SEQ ID NO:22, one GATA-1 binding site having the nucleotide sequenceof TTATCT is located at position 173 to position 178, one GATA-1 bindingsite having the nucleotide sequence of CTATCA located at position 210 toposition 215, and one binding site for EKLF having the nucleotidesequence of CCACACACA is located at position 183 to position 191.

In another non-limiting embodiment, the 286 bp core sequence of humanHS1 has the nucleotide sequence set forth in SEQ ID NO:23 providedbelow:

[SEQ ID NO: 23] CTGAGCAACTAATCATGCAGGACTCTCAAACACTAACCTATAGCCTTTTCTATGTATCTACTTGTGTAGAAACCAAGCGTGGGGACTGAGAAGGCAATAGCAGGAGCATTCTGACTCTCACTGCCTTTAGCTAGGCCCCTCCCTCATCACAGCTCAGCATAGTCCTGAGCTCTTATCTATATCCACACACAGTTTCTGACGCTGCCCAGCTATCACCATCCCAAGTCTAAAGAAAAAAATAATGGGTTTGCCCATCTCTGTTGATTAGAAAACAAAACAAAATAAA

The nucleotide sequence set forth in SEQ ID NO:23 corresponds tonucleotides position 21481 to position 21766 of SEQ ID NO:19 (GenBankAccess No.: NG_000007.3). In SEQ ID NO:23, one GATA-1 binding sitehaving the nucleotide sequence of TTATCT is located at position 173 toposition 178, one GATA-1 binding site having the nucleotide sequence ofCTATCA located at position 210 to position 215, and one binding site forEKLF having the nucleotide sequence of CCACACACA is located at position183 to position 191.

In certain embodiments, the β-globin LCR region does not comprise a HS1region that comprises a core sequence of HS1. A HS1 region thatcomprises a core sequence of HS1 can vary in length and sequence. Innon-limiting examples, a HS1 region that comprises a core sequence ofHS1 is from about 300 bp to about 1200 bp, e.g., from about 300 bp toabout 400 bp, from about 400 bp to about 500 bp, from about 500 bp toabout 600 bp, from about 600 bp to about 700 bp, from about 700 bp toabout 800 bp, from about 800 bp to about 900 bp, from about 900 bp toabout 1000 bp, from about 1000 bp to about 1100 bp, or from about 1100bp to about 1200 bp, in length. In one non-limiting embodiment, theβ-globin LCR region does not comprise an about 1.0 kb bp HS1 region. Inone non-limiting embodiment, the β-globin LCR region does not comprisean about 1.1 kb HS1 region.

In certain embodiments, the β-globin LCR region does not comprise a HS1region that sustains the activity of HS1, e.g., enhancer activity, orfunctioning as a facilitator or regulatory element to tether theenhancer activity of other HS regions, e.g., HS2-4. In certainembodiments, the β-globin LCR region does not comprise a HS1 region thatis capable of enhancing the transcription of a globin gene (e.g., humanβ-globin gene). In non-limiting examples, the β-globin LCR region doesnot comprise a HS1 region whose ability to enhance the transcription ofa globin gene (e.g., human β-globin gene) is no less than 60%, no lessthan 70%, no less than 80%, no less than 90%, or no less than 95% incomparison to a native HS1 region. In non-limiting examples, theβ-globin LCR region does not comprise a HS1 region whose ability totether the enhancer activity of one or more of HS2-HS4 is no less than60%, no less than 70%, no less than 80%, no less than 90%, or no lessthan 95% in comparison to a native HS1 region.

In certain embodiments, the β-globin LCR region does not comprise a HS1region that comprises one, two, or three of the following binding sites:two GATA-1 binding sites (e.g., TTATCT, and CTATCA), and one bindingsite for EKLF (e.g., CCACACACA). In certain embodiments, the β-globinLCR region does not comprise a HS1 region that comprises two of theabove-described binding sites. For example, in certain embodiments, theβ-globin LCR region does not comprise a HS1 region that comprises twoGATA-1 binding sites and does not comprise one binding site for EKLF. Incertain embodiments, the β-globin LCR region does not comprise a HS1region that comprises one not two AP1/NF-E2 binding site and one bindingsite for EKLF.

In certain embodiments, the β-globin LCR region comprises a HS3 regionand a HS4 region, and the β-globin LCR region does not comprise a HS1region or a HS2 region. In certain embodiments, the HS3 region and HS4region within the β-globin LCR region are contiguous. In onenon-limiting embodiment, the β-globin LCR region consisting essentiallyof a HS3 region and a HS4 region. In another embodiment, the β-globinLCR region comprises two introduced GATA-1 binding sites at the junctionbetween the HS3 region and the HS4 region. The HS3 region can liebetween the globin gene or functional portion thereof and the HS4region.

In certain embodiments, the β-globin LCR region comprises a HS3 regionhaving the nucleotide sequence set forth in SEQ ID NO:5 and a HS4 regionhaving the nucleotide sequence set forth in SEQ ID NO:6, SEQ ID NO:7 orSEQ ID NO:8, and the β-globin LCR region does not comprise a HS1 regionor a HS2 region.

In one non-limiting embodiment, the β-globin LCR region comprises a HS3region having the nucleotide sequence set forth in SEQ ID NO:5 and a HS4region having the nucleotide sequence set forth in SEQ ID NO:6, and theβ-globin LCR region does not comprise a HS1 region or a HS2 region, asshown in FIG. 4.

Globin Gene

In accordance with the presently disclosed subject matter, theexpression cassette comprises a globin gene or a functional portionthereof. The globin gene can be a β-globin gene, a γ-globin gene, or aδ-globin gene. In certain embodiments, the expression cassette comprisesa human β-globin gene. In accordance with the presently disclosedsubject matter, the human β-globin gene can be a wild-type humanβ-globin gene, a deleted human β-globin gene comprising one or moredeletions of intron sequences, or a mutated human β-globin gene encodingat least one anti-sickling amino acid residue. In one non-limitingembodiment, a presently disclosed expression cassette comprises awild-type human β-globin gene. In another embodiment, the a presentlydisclosed expression cassette comprises a human β^(A)-globin geneencoding a threonine to glutamine mutation at codon 87 (β^(A-T87Q)). Theglutamine residue at position 87 in the gamma-globin chain augments theanti-sickling activity of the gamma chain relative to the beta chain,while preserving adult oxygen-binding characteristics of the beta chain(Nagel et al., Proc. Natl. Acad. Sci. U.S.A. (1979); 76:670-672). Incertain embodiments, a functional portion of a globin gene has at least80%, at least 90%, at least 95%, or at least 99% identity to acorresponding wild-type reference polynucleotide sequence.

Promoters and Enhancers

In accordance with the presently disclosed subject matter, theexpression cassette can further comprise a β-globin promoter. In certainembodiments, the β-globin promoter is positioned between the globin geneor functional portion thereof and the β-globin LCR region. The lengthand the sequence of the β-globin promoter can vary. In certainembodiments, the β-globin promoter is from about 100 bp to about 1600 bpin length, e.g., from about 200 bp to about 700 bp, from about 100 bp toabout 200 bp, from about 200 bp to about 300 bp, from about 300 bp toabout 400 bp, from about 400 bp to about 500 bp, from about 500 bp toabout 600 bp, from about 600 bp to about 700 bp, from about 700 bp toabout 800 bp, from about 800 bp to about 900 bp, from about 900 bp toabout 1000 bp, from about 1000 bp to about 1100 bp, from about 1100 bpto about 1200 bp, from about 1200 bp to about 1300 bp, from about 1300bp to about 1400 bp, from about 1400 bp to about 1500 bp, or from about1500 bp to about 1600 bp in length. In certain embodiments, the β-globinpromoter a human β-globin promoter that is about 130 bp, about 613 bp,about 265 bp, or about 1555 bp, in length. In one embodiment, theβ-globin promoter is a human β-globin promoter that is about 613 bp inlength. In one non-limiting example, the human β-globin promoter has thenucleotide sequence set forth in SEQ ID NO:10, which is provided below:

[SEQ ID NO: 10] AAGCAATAGATGGCTCTGCCCTGACTTTTATGCCCAGCCCTGGCTCCTGCCCTCCCTGCTCCTGGGAGTAGATTGGCCAACCCTAGGGTGTGGCTCCACAGGGTGAGGTCTAAGTGATGACAGCCGTACCTGTCCTTGGCTCTTCTGGCACTGGCTTAGGAGTTGGACTTCAAACCCTCAGCCCTCCCTCTAAGATATATCTCTTGGCCCCATACCATCAGTACAAATTGCTACTAAAAACATCCTCCTTTGCAAGTGTATTTACGTAATATTTGGAATCACAGCTTGGTAAGCATATTGAAGATCGTTTTCCCAATTTTCTTATTACACAAATAAGAAATTGATGCACTAAAAGTGGAAGAGTTTTGTCTACCATAATTCAGCTTTGGGATATGTAGATGGATCTCTTCCTGCGTCTCCAGAATATGCAAAATACTTACAGGACAGAATGGATGAAAACTCTACCTCAGTTCTAAGCATATCTTCTCCTTATTTGGATTAAAACCTTCTGGTAAGAAAAGAAAAAAAATATATATATATATGTGTATATATACACACATACATATACATATATATGCATTCATTTGTTGTTGTTTTTCT TAATTTGCTCATG

In one embodiment, the β-globin promoter is a human β-globin promoterthat is about 265 bp in length. In one non-limiting example, the humanβ-globin promoter has the nucleotide sequence set forth in SEQ ID NO:11.

[SEQ ID NO: 11] AAGCAATAGATGGCTCTGCCCTGACTTTTATGCCCAGCCCTGGCTCCTGCCCTCCCTGCTCCTGGGAGTAGATTGGCCAACCCTAGGGTGTGGCTCCACAGGGTGAGGTCTAAGTGATGACAGCCGTACCTGTCCTTGGCTCTTCTGGCACTGGCTTAGGAGTTGGACTTCAAACCCTCAGCCCTCCCTCTAAGATATATCTCTTGGCCCCATACCATCAGTACAAATTGCTACTAAAAACATCCTCCTT TGCAAGTGTATTTAC

Additionally or alternatively, a presently disclosed expression cassettecan further comprise a human β-globin 3′ enhancer. In certainembodiments, the human β-globin 3′ enhancer is positioned in theupstream of the globin gene or functional portion thereof. In certainembodiments, the β-globin 3′ enhancer is from about 500 bp to about 1000bp in length, e.g., from about 500 bp to about 600 bp, from about 600 bpto about 700 bp, from about 700 bp to about 800 bp, or from about 800 bpto about 900 bp in length. In one embodiment, the human β-globin 3′enhancer is about 879 bp in length. In one example, the human β-globin3′ enhancer has the nucleotide sequence set forth in SEQ ID NO:12.

[SEQ ID NO: 12] TAGGTATTGAATAAGAAAAATGAAGTTAAGGTGGTTGATGGTAACACTATGCTAATAACTGCAGAGCCAGAAGCACCATAAGGGACATGATAAGGGAGCCAGCAGACCTCTGATCTCTTCCTGAATGCTAATCTTAAACATCCTGAGGAAGAATGGGACTTCCATTTGGGGTGGGCCTATGATAGGGTAATAAGACAGTAGTGAATATCAAGCTACAAAAAGCCCCCTTTCAAATTCTTCTCAGTCCTAACTTTTCATACTAAGCCCAGTCCTTCCAAAGCAGACTGTGAAAGAGTGATAGTTCCGGGAGACTAGCACTGCAGATTCCGGGTCACTGTGAGTGGGGGAGGCAGGGAAGAAGGGCTCACAGGACAGTCAAACCATGCCCCCTGTTTTTCCTTCTTCAAGTAGACCTCTATAAGACAACAGAGACAACTAAGGCTGAGTGGCCAGGCGAGGAGAAACCATCTCGCCGTAAAACATGGAAGGAACACTTCAGGGGAAAGGTGGTATCTCTAAGCAAGAGAACTGAGTGGAGTCAAGGCTGAGAGATGCAGGATAAGCAAATGGGTAGTGAAAAGACATTCATGAGGACAGCTAAAACAATAAGTAATGTAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTACTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGCATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTATGTTTTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACATCATTG

Furthermore, a presently disclosed expression cassette can furthercomprise at least one erythroid-specific enhancer. The presentlydisclosed expression cassette allows for expression of a globin gene(e.g., human β-globin gene) in erythroid-specific fashion. Theerythroid-specific enhancer can enhance the expression of the globingene in erythroid-specific fashion. For example, the erythroid-specificenhancer lack enhancer activity in non-erythroid tissues. Inparticularly, for the β-globin LCR region that lacks a HS2 region, whichprimarily functions as an expression enhancer, the addition of one ormore erythroid-specific enhancers can compensate the enhancing activityof a HS2 region. Furthermore, the presently disclosed erythroid-specificenhancers do not decrease or reduce the titer of a vector comprising theexpression cassette. The length of the erythroid-specific enhancer canvary, e.g., from about 100 bp to about 200 bp, from about 100 bp toabout 120 bp, from about 120 bp to about 140 bp, from about 140 bp toabout 200 (e.g., from about 140 bp to about 150 bp, from about 150 bp toabout 160 bp, from about 160 bp to about 170 bp, from about 170 bp toabout 180 bp, from about 180 bp to about 190 bp, or from about 190 bp toabout 200 bp). In certain embodiments, the erythroid-specific enhancerhas a length of from about 140 bp to about 200 bp. In one non-limitingembodiment, the erythroid-specific enhancer has a length of 152 bp,which has the nucleotide sequence set forth in SEQ ID NO:13, which isprovided below:

[SEQ ID NO: 13] TCTCCCACGCCCTGGTCTCAGCTTGGGGAGTGGTCAGACCCCAATGGCGATAAACTCTGGCAACTTTATCTGTGcaCTGCAGGCTCAGCCCCAAcaGCTTTAGCTTTCACAAGCAGGCAGGGGAAGGGAAACACATATCTCCAGATATGA GG

In one non-limiting embodiment, the erythroid-specific enhancer has alength of 157 bp, which has the nucleotide sequence set forth in SEQ IDNO:14, which is provided below:

[SEQ ID NO: 14] CTAAACCCCTCCCCCACCCTAGCCCCAAGCTTCATCTTAGCTCCACTCCTGACCCTATCCAGCTAAAGGTCCCCACCCAGCTCCTGCCTATCTAGTCATTGCATATGGCAAGACTTGAAAGTCCTATCTCAAAGCAGCAGAATTATCAGC TACGACT

In one non-limiting embodiment, the erythroid-specific enhancer has alength of 141 bp, which has the nucleotide sequence set forth in SEQ IDNO:15, which is provided below:

[SEQ ID NO: 15] CCATCCCCCAGCACTCCCTGCCCCCACAGCCCAGACTTGACCAACTCCCAGCTccGCCTGGGACTTCCAGATATGGGGCCCCACCCTTGCAGGCCTTGGGGACGCTGAAGATATTGACTATCTGCGTGCCggAAAAGGGTG

In one non-limiting embodiment, the erythroid-specific enhancer has alength of 171 bp, which has the nucleotide sequence set forth in SEQ IDNO:16, which is provided below:

[SEQ ID NO: 16] AAAGGCTGGGGGTGGGAGTAGCGGATTTGAAGCACTTGTTGGCCTACAGAGGTGTGGCAAGCAGAGCACCTCAGAACTCAGGCGTACTGCCCGCCGCCCGAGCCCTGCGAGGGCCGATAGCGAGGGTGTGGCCCTTATCTGCACCCAGCA GAGCGCCGGCGGGGTACGGTC

In one non-limiting embodiment, the erythroid-specific enhancer has alength of 195 bp, which has the nucleotide effluence set forth in SRO TDNO. 17 which is provided below:

[SEQ ID NO: 17] CAGTTGCCTCAGCTGAGTATGTCTTCTAAAGATAATGTCGATTGTGTATGGCTGATGGGATTCTAGGACCAAGCAAGAGGTTTTTTTTTTTCCCCCACATACTTAACGTTTCTATATTTCTATTTGAATTCGACTGGACAGTTCCATTTGAATTATTTCTCTCTCTCTCTCTCTCTGACACATTTTATCTTGCCA

Erythroid-specific enhancers can be identified and determined by anysuitable methods known in the art. The erythroid-specific enhancers canbe positioned at the 3′ LTR (downstream) or the 5′ LTR (downstream) ofthe β-globin LCR region. In one embodiment, the at least oneerythroid-specific enhancer is positioned in the 5′ LTR of the β-globinLCR region, e.g., the upstream of the HS3 region. The expressioncassette can comprise one, two, three, four, or five erythroid-specificenhancers. In one embodiment, the expression cassette comprises oneerythroid-specific enhancer. In another embodiment, the expressioncassette comprises two erythroid-specific enhancers. In yet anotherembodiment, the expression cassette comprises three erythroid-specificenhancers. In certain embodiments, the expression cassette comprisesfour erythroid-specific enhancers. In a non-limiting embodiment, theexpression cassette comprises five erythroid-specific enhancers.

Insulators

In accordance with the presently disclosed subject matter, theexpression cassette comprises at least one of the above-describedinsulators. In certain embodiments, a presently disclosed expressioncassette comprises at least one insulator comprising the CTCF bindingsite sequence set forth in SEQ ID NO:18, for example, but not limitedto, an insulator comprising SEQ ID NO: 24 or SEQ ID NO:25, such as aninsulator having the nucleotide sequence set forth in SEQ ID NO:1 (i.e.,insulator A1). In various non-limiting embodiments, the insulator can beincorporated or inserted into one or both LTRs or elsewhere in theregion of a presently disclosed expression cassette that integrates intothe cellular genome. In one embodiment, the insulator is positioned atthe 3′ end of the expression cassette. In one embodiment, the insulatoris positioned at the 5′ end of the expression cassette. In oneembodiment, the expression cassette comprises two of the insulatorhaving the nucleotide sequence set forth in SEQ ID NO:1, where oneinsulator is positioned at the 3′ end and the other insulator ispositioned at the 5′ end of the expression cassette.

The presently disclosed insulators possess powerful enhancer blockingactivity. In certain embodiments, the insulators possess barrieractivity in addition to enhancer blocking activity. The presentlydisclosed insulators substantially decrease the risks of insertionalmutagenesis and genotoxicity associated with viral vectors. Furthermore,when a presently disclosed insulator is incorporated into a vector, theinsulator does not adversely effect vector titers of the vector. Incertain embodiments, the insulators (e.g., insulator A1) increase the invivo expression of the globin gene or functional portion thereof. Forthe purpose of illustration and not limitation, FIGS. 1-4 showrecombinant vectors comprising exemplary expression cassettes inaccordance with certain embodiments of the presently disclosed subjectmatter. FIG. 1 shows a recombinant vector comprising a presentlydisclosed expression cassette that comprises a human β^(A-T87Q) globingene, which is operably linked to a β-globin LCR region that comprises a860 bp HS2 region (e.g., one having the nucleotide sequence set forth inSEQ ID NO:9), a 1301 bp HS3 region (e.g., one having the nucleotidesequence set forth in SEQ ID NO:5), and a 1065 bp HS4 region (e.g., onehaving the nucleotide sequence set forth in SEQ ID NO:7).

FIG. 2 shows one exemplary recombinant vector comprising an expressioncassette in accordance with one embodiment of the presently disclosedsubject matter. FIG. 2 shows a recombinant vector comprising a presentlydisclosed expression cassette that comprises a human β^(A-T87Q) globingene, which is operably linked to a β-globin LCR region that comprises a1.1 kb HS1 region (e.g., one having the nucleotide sequence set forth inSEQ ID NO:2), a 1301 bp HS3 region (e.g., one having the nucleotidesequence set forth in SEQ ID NO:5), and a 1065 bp HS4 region (e.g., onehaving the nucleotide sequence set forth in SEQ ID NO:6).

FIG. 3 shows one exemplary recombinant vector comprising an expressioncassette in accordance with one embodiment of the presently disclosedsubject matter. FIG. 3 shows a recombinant vector comprising a presentlydisclosed expression cassette that comprises a human β^(A-T87Q) globingene, which is operably linked to a β-globin LCR region that comprises a602 bp HS1 region (e.g., one having the nucleotide sequence set forth inSEQ ID NO:3), a 1301 bp HS3 region (e.g., one having the nucleotidesequence set forth in SEQ ID NO:5), and a 446 bp HS4 region (e.g., onehaving the nucleotide sequence set forth in SEQ ID NO:8).

FIG. 4 shows one exemplary recombinant vector comprising an expressioncassette in accordance with one embodiment of the presently disclosedsubject matter. FIG. 4 shows a recombinant vector comprising a presentlydisclosed expression cassette that comprises a human β^(A-T87Q) globingene, which is operably linked to a β-globin LCR region that comprises a1301 bp HS3 region (e.g., one having the nucleotide sequence set forthin SEQ ID NO:5), and a 1065 bp HS4 region (e.g., one having thenucleotide sequence set forth in SEQ ID NO:6). The expression cassetteshown in FIG. 4 also comprises the following five erythroid-specificenhancers (shown as “EE5” in FIG. 4): one erythroid-specific enhancerhaving the nucleotide sequence set forth in SEQ ID NO:13, oneerythroid-specific enhancer having the nucleotide sequence set forth inSEQ ID NO:14, one erythroid-specific enhancer having the nucleotidesequence set forth in SEQ ID NO:15, one erythroid-specific enhancerhaving the nucleotide sequence set forth in SEQ ID NO:16, and oneerythroid-specific enhancer having the nucleotide sequence set forth inSEQ ID NO:17.

As shown in FIGS. 1-4, each of the expression cassettes comprise aninsulator having the nucleotide sequence set forth in SEQ ID NO:1 (i.e.,insulator A1). In addition, as shown in FIGS. 1-4, each of theexpression cassettes comprise a 879 bp human β-globin 3′ enhancer, whichis positioned upstream of the human β-globin gene. Furthermore, as shownin FIGS. 1-4, each of the recombinant vectors comprise a Woodchuckhepatitis post-regulatory element (WPRE) and a bovine growth hormonepolyadenylation signal in the 3′ long terminal repeat (LTR) of thevector (e.g., 3′ to the R region in the 3′ LTR).

III. VECTORS, NUCLEASES AND CRISPR-CAS SYSTEMS

The presently disclosed subject matter provides vectors and deliverysystems (e.g., a non-naturally occurring or engineered nucleases or aCRISPR-Cas system) comprising the above-described expression cassettes.The vectors and delivery systems are suitable delivery vehicles for thestable introduction of globin gene (e.g., human β-globin) into thegenome of a broad range of target cells to increase expression of theglobin protein (human β-globin protein) in the cell.

In certain embodiments, the vector is a retroviral vector (e.g., gammaretroviral or lentiviral) that is employed for the introduction ortransduction of the above-described expression cassette into the genomeof a host cell (e.g., a hematopoietic stem cell, an embryonic stem cell,an induced pluripotent stem cell, or a hemogenic endothelium cell). Incertain embodiments, the retroviral vector comprises an expressioncassette that comprises one of the above-described insulators, e.g.,insulator A1. The insulator can be positioned at the 3′ or the 5′ end ofthe expression cassette. In one embodiment, the insulator is positionedat the 3′ end of the expression cassette. During reverse transcriptionand vector integration, the insulator positioned at the 3′ end is copiedinto the 5′ end of the expression cassette. The resulting topologyplaces copies of the insulator between the genomic regions located atthe 5′ LTR and the 3′ LTR of the integrated virus and enhancer activityfrom the 5′ LTR and internal package promoter, but does not contain theenhancer in the 3′ LTR. This topology can decrease genotoxicity, therebyresulting in decreased tumor formation and increased survival of theanimals.

In certain embodiments, the recombinant vector further comprises aWoodchuck hepatitis post-regulatory element (WPRE) in the 3′ longterminal repeat (LTR) of the vector (e.g., 3′ to the R region in the 3′LTR of the vector). In certain embodiments, the recombinant vectorfurther comprises a bovine growth hormone polyadenylation signal inaddition to the WPRE in the 3′ long terminal repeat (LTR) of the vector(e.g., 3′ to the R region in the 3′ LTR of the vector). An essentialfeature of therapeutic globin vectors is to achieve a high titer,sufficient for effective transduction of patient cells. By virtue oftheir large cargo, comprising a gene, promoter, enhancers and/or LCRelements, globin lentiviral vectors inherently have low titer,complicating their manufacture and limiting their clinical use. Thisproblem is further compounded by the incorporation of additional genomicelements such as an insulator, which further increase the size of thevector. The WPRE can increase the titer of the recombinant vector.Addition of a bovine growth hormone polyadenylation signal to the WPREcan further increase the titer of the recombinant vector. In certainembodiments, the WPRE and the bovine growth hormone polyadenylationsignal are not comprised within the expression cassette, and thus, nottransferred to the cells transduced with the recombinant vector. Theincorporation of these elements for enhancing the production of globinlentiviral vectors is essential to yield higher titers and hence for theclinical usefulness of the vectors described in this application.

In one non-limiting example, a presently disclosed expression cassettecan be cloned into a retroviral vector and expression can be driven fromits endogenous promoter, from the retroviral long terminal repeat, orfrom an alternative internal promoter. Combinations of retroviral vectorand an appropriate packaging line are also suitable, where the capsidproteins will be functional for infecting human cells. Variousamphotropic virus-producing cell lines are known, including, but notlimited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437);PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP(Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464).Non-amphotropic particles are suitable too, e.g., particles pseudotypedwith VSVG, RD114 or GALV envelope and any other known in the art.

Suitable methods of transduction also include direct co-culture of thecells with producer cells, e.g., by the method of Bregni, et al. (1992)Blood 80:1418-1422, or culturing with viral supernatant alone orconcentrated vector stocks with or without appropriate growth factorsand polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat.22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

Transducing viral vectors can be used to express a globin gene (e.g., ahuman β-globin gene) in a host cell (e.g., hematopoietic stem cells, anembryonic stem cell, or an induced pluripotent stem cell). Preferably,the chosen vector exhibits high efficiency of infection and stableintegration and expression (see, e.g., Cayouette et al., Human GeneTherapy (1997); 8:423-430; Kido et al., Current Eye Research (1996);15:833-844; Bloomer et al., Journal of Virology (1997); 71:6641-6649;Naldini et al., Science (1996); 272:263 267; and Miyoshi et al., Proc.Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors that can beused include, for example, adenoviral, lentiviral, and adeno-associatedviral vectors, vaccinia virus, a bovine papilloma virus, or a herpesvirus, such as Epstein-Barr Virus (also see, for example, the vectors ofMiller, Human Gene Therapy (1990); 15-14; Friedman, Science (1989); 244:1275-1281; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev etal., Current Opinion in Biotechnology (1990); 1:55-61; Sharp, The Lancet(1991); 337:1277-1278; Cornetta et al., Nucleic Acid Research andMolecular Biology (1987)36:311-322; Anderson, Science (1984);226:401-409; Moen, Blood Cells (1991); 17:407-416; Miller et al.,Biotechnology (1989); 7:980-990; Le Gal La Salle et al., Science (1993);259:988-990; and Johnson, Chest (1995); 107:775-83S). Retroviral vectorsare particularly well developed and have been used in clinical settings(Rosenberg et al., N. Engl. J. Med (1990); 323:370; Anderson et al.,U.S. Pat. No. 5,399,346).

The requirement for efficient delivery and integration make retroviralvectors suitable for transducing a presently disclosed expressioncassette. Retroviral vectors can be derived from three genera of theretroviridae: the γ-retroviruses (also known as C-type murineretroviruses or oncoretroviruses), the lentiviruses, and thespumaviruses (also known as foamy viruses). Several reviews detailingmolecular approaches for the generation of replication-defectiveretroviral particles are available (Cornetta et al. (2005); Cockrell &Kafri (2007)). The vector itself, which encodes the therapeutictransgene or cDNA, retains the minimal viral sequences needed to enablepackaging in viral particles in a packaging cell line, reversetranscription, and integration. The packaging cell expresses thenecessary structural proteins and enzymes that are required to assemblean infectious recombinant particle that contains the vector sequence andthe machinery needed for its reverse transcription and integration inthe transduced cell.

While the manufacturing aspects of all retroviral vector types followthe same general principles, γ-retroviral, lentiviral and spumaviralvectors differ in some of their intrinsic biological properties.Gamma-retroviruses, including the prototypic murine leukaemia viruses(MLV), effectively infect many cell types but are unable to integrate incells that do not proceed to S phase soon after infection. In contrast,lentiviruses and their vector derivatives can transduce nondividingcells (Follenzi & Naldini, 2002; Salmon & Trono, 2002) owing to theirability to translocate to the nucleus and integrate in the absence ofcell division (Lewis & Emerman, 1994; Goff, 2001). Another fundamentalattribute of lentiviral vectors is their relative genomic stability, asestablished for globin lentiviral vectors (May et al., 2000), whichcontrasts with the genomic instability of MLV-based globin vectors(Leboulch et al., 1994; Sadelain et al., 1995). Lentiviral and foamyvectors further provide a greater packaging capacity (Kumar et al.,2001; Rethwilm, 2007). All three vector types have been usedsuccessfully for the transduction of cytokineactivated HSCs (Miyoshi etal., 1999; Josephson et al., 2002; Leurs et al., 2003).

These three vector systems differ in their integration patterns. Theintegration pattern of retroviruses is semi-random and biased towardsgenes and their vicinity in approximately two-thirds of all integrationevents (Schroder et al., 2002; Wu et al., 2003; Mitchell et al., 2004;De Palma et al., 2005; Trobridge et al., 2006). There are however subtleand possibly significant differences in their exact distribution.Gamma-retroviruses have a propensity to integrate upstream oftranscribed genes, whereas lentiviruses and lentiviral vectors targetthe entire transcribed gene sequence. Foamy vectors appear to be lessprone to intragenic integration (Trobridge et al., 2006). In oneembodiment, the vector comprising the expression cassette is alentivirus vector. The vectors can be derived from humanimmunodeficiency-1 (HIV-1), human immunodeficiency-2 (HIV-2), simianimmunodeficiency virus (SIV), feline immunodeficiency virus (FIV),bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV),equine infectious anemia virus (EIAV), caprine arthritis encephalitisvirus (CAEV) and the like. In one non-limiting embodiment, thelentiviral vector is an HIV vector. HIV-based constructs are the mostefficient at transduction of human cells.

The semi-random pattern of vector integration exposes patients to therisk of insertional oncogenesis when the vector trans-activates aneighboring oncogene. This may result in clonal expansion (Ott et al,2006; Cavazzana-Calvo et al, 2010), myelodysplasia (Stein et al, 2010)or leukaemia (Hacein-Bey-Abina et al, 2003, 2008; Howe et al, 2008).Targeted gene delivery strategies, utilizing a non-naturally occurringor engineered nuclease (including, but not limited to, Zinc-fingernuclease (ZNFs), meganuclease, transcription activator-like effectornuclease (TALEN)), or a CRISPR-Cas system, can reduce or even eliminatethe concern of insertional oncogenesis that is inherent to the use ofretroviral vectors.

Eukaryotic cells utilize two distinct DNA repair mechanisms in responseto DNA double strand breaks (DSBs): Homologous recombination (HR) andnon-homologous end-joining (NHEJ). The activation of the HR repairmachinery depends on the cell cycle status, and it is restricted to theS and G2 phases; in contrast, the NHEJ pathway is active throughout thecell cycle. Mechanistically, HR is an error-free DNA repair mechanism,because it requires a homologous template to repair the damaged DNAstrand. On the other hand, NHEJ is a template-independent repairmechanism that is imprecise, due to DNA end processing during repairthat leads to insertions or deletions at the DNA break site (Moynahan &Jasin, 2010). Because of its homology-based mechanism, HR has been usedas a tool to site-specifically engineer the genome of different species.From a therapeutic perspective, HR has been successfully used to repairmutated genes, thus offering a promising approach to cell-mediatedtreatment of monogenic diseases (Porteus et al, 2006).

Gene targeting by HR requires the use of two homology arms that flankthe transgene/target site of interest. Generally, standard plasmid DNAshave been used to deliver 5-10 kb homology arms along with transgenesfor positive and negative selection. This method is commonly used toknock-out/knockin genes in mouse embryonic stem (mES) cells (Capecchi,2005; FIG. 2B). In human cells, the use of this approach has allowedgene targeting with efficiencies in the order of 10⁻⁶, which are lowerthan in mES cells and are not therapeutically practical. HR efficiencycan be increased by the introduction of DNA-doubled stranded breaks(DSBs) at the target site using specific rare-cutting endonucleases,resulting in over 1,000-fold increase in correct gene targeting (Jasin,1996). The discovery of this phenomenon prompted the development ofmethods to create site-specific DSBs in the genome of different species.Various chimeric enzymes have been designed for this purpose over thelast decade, namely zinc-finger nucleases (ZFNs), meganucleases, andtranscription activator-like effector nucleases (TALENs).

ZFNs are modular chimeric proteins that contain a ZF-based DNA bindingdomain (DBD) and a FokI nuclease domain (Porteus & Carroll, 2005). DBDis usually composed of three ZF domains, each with 3-base pairspecificity; the FokI nuclease domain provides a DNA nicking activity,which is targeted by two flanking ZFNs. Owing to the modular nature ofthe DBD, any site in a genome could be targeted in principle. However,as a single ZFN can bind and nick DNA, there is potential for a highnumber of off-target effects, resulting in the activation of the NHEJpathway that may either introduce insertions/deletions or integrate thetargeting vector in a non-specific manner. Obligate FokI domains thatcan nick their respective DNA strand only when they form a heterodimerwere recently reported (Doyon et al, 2011). The use of such obligateZFNs can reduce the genotoxic effects of this approach.

Meganucleases (MNs)/homing endonucleases (HEs) are dsDNA nucleases thatrecognize and cleave large DNA sites (14-40 bp) with low cleavagefrequencies in eukaryotic genomes (Paques & Duchateau, 2007). Althoughthis limits the potential target sites, MN-DNA structures have been usedas a guide to specifically modify DNA-interacting residues in order tochange the MN specificity (Marcaida et al, 2010). I-CreI has beensuccessfully engineered to generate chimeric meganucleases that targetthe human XPC and RAG1 genes, and they have been shown to stimulate HRactivity in mammalian cells with no evident genotoxicity (Redondo et al,2008; Grizot et al, 2009). The genotoxicity of this approach will needto be compared to that of ZFNs and TALE nucleases.

TALENs are similar ZFN except that the DBD is derived from transcriptionactivator-like effcetors (TALEs), which are virulent factors used byphytopathogenic bacteria (Herbers, 1992). The TALE DBD is modular, andit is composed of 34-residue repeats, and its DNA specificity isdetermined by the number and order of repeats (Herbers, 1992). Eachrepeat binds a single nucleotide in the target sequence through only tworesidues (Boch, 2011). The advantage over ZFN technology is the rapidconstruction of DBDs.

A number of studies have used these chimeric enzymes to stimulate HR foreither gene addition or gene repair at their target site (Paques &Duchateau, 2007; Urnov et al, 2010). Porteus designed a ZFN to a halfsite sequence from the human HBB that surrounds the sickle cell mutationnucleotide (Porteus, 2006). This ZFN targets the sequence and stimulatesHR at a chimeric DNA target when combined with a ZFN targeting theZif268 binding site. There have been recent advances in targeting genesin cord blood CD34⁺ cells. Use of non-integrating lentiviruses todeliver ZFNs and the donor DNA in these cells to target the CCR5 genewas reported in Lombardo et al, 2007. Lombardo et al, 2007 showed geneaddition at this locus with correct targeting in 80% of the positivelyselected cells.

The presently disclosed subject matter provides a non-naturallyoccurring or engineered nuclease comprising a presently disclosedexpression cassette, as described above. Suitable nucleases include, butare not limited to, ZFNs, meganucleases, and TALENs. A presentlydisclosed nuclease comprises a DNA binding domain and a nucleasecleavage domain. The DNA binding domain of the nuclease can beengineered to bind to a sequence of choice, e.g., a predetermined site.An engineered DNA binding domain can have a distinct bindingspecificity, compared to a naturally occurring nuclease. Engineeringmethods include, but are not limited to, rational design and varioustypes of selection. Any suitable cleavage domain can be operativelylinked to a DNA-binding domain to form a nuclease. For example,Zinc-finger protein (ZFP) DNA-binding domains can be fused to nucleasecleavage domains to create ZFNs-a functional entity that is able torecognize its intended nucleic acid target through its engineered ZFPDNA binding domain and cause the DNA to be cut near the ZFP binding sitevia the nuclease activity. See, e.g., Kim et al. Proc Nat'l Acad Sci USA(1996); 93(3):1156-1160. Likewise, TALE DNA-binding domains can be fusedto nuclease cleavage domains to create TALENs. See, e.g., U.S.Publication No. 20110301073.

The cleavage domain can be heterologous to the DNA-binding domain, e.g.,a meganuclease DNA-binding domain and cleavage domain from a differentnuclease. Heterologous cleavage domains can be obtained from anyendonuclease or exonuclease. Exemplary endonucleases from which acleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalog, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional regions thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from theabove-described nuclease that requires dimerization for cleavageactivity. In general, two fusion proteins are required for cleavage ifthe fusion proteins comprise cleavage half-domains. Alternatively, asingle protein comprising two cleavage half-domains can be used. The twocleavage half-domains can be derived from the same endonuclease (orfunctional portions thereof), or each cleavage half-domain can bederived from a different endonuclease (or functional portions thereof).

In certain embodiments, the nuclease comprises an expression cassettethat comprises two of the above-described insulators, e.g., two of theinsulator having the nucleotide sequence set forth in SEQ ID NO:1. Oneof the two insulators is positioned at the 3′ end of the expressioncassette, and the other insulator is positioned at the 5′ end of theexpression cassette.

The presently disclosed subject matter also provides a non-naturallyoccurring or engineer CRISPR-Cas system comprising the above-describedexpression cassette. The CRISPR (Clustered Regularly Interspaced ShortPalindromic Repeats)-Cas (CRISPR Associated) system is an engineerednuclease system based on a bacterial system that can be used for genomeengineering. It is based on part of the adaptive immune response of manybacteria and archea. When a virus or plasmid invades a bacterium,segments of the invader's DNA are converted into CRISPR RNAs (crRNA) bythe “immune” response. The crRNA then associates, through a region ofpartial complementarity, with another type of RNA called tracrRNA toguide a CRISPR-Cas nuclease to a region homologous to the crRNA in thetarget DNA called a “proto spacer”. The CRISPR-Cas nuclease cleaves theDNA to generate blunt ends at the DSB at sites specified by a20-nucleotide guide sequence contained within the crRNA transcript. TheCRISPR-Cas nuclease requires both the crRNA and the tracrRNA for sitespecific DNA recognition and cleavage. This system has been engineeredsuch that the crRNA and tracrRNA can be combined into one molecule (the“single guide RNA”); and the crRNA equivalent portion of the singleguide RNA can be engineered to guide the CRISPR-Cas nuclease to targetany desired sequence (see Jinek et al., Science (2012); 337:816-821).Thus, the CRISPR-Cas system can be engineered to create a DSB at adesired target in a genome. In certain embodiments, the CRISPR-Cassystem comprises a CRISPR-Cas nuclease and a single-guide RNA. Suitableexamples of CRISPR-Cas nucleases include, but are not limited to, Cas1,Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known asCsn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. TheseCRISPR-Cas nucleases are known; for example, the amino acid sequence ofS. pyogenes Cas9 protein may be found in the SwissProt database underaccession number Q99ZW2. In some embodiments, the CRISPR-Cas nucleasehas DNA cleavage activity, e.g., Cas9. In certain embodiments, theCRISPR-Cas nuclease is Cas9. The CRISPR-Cas nuclease can direct cleavageof one or both strands at the location of a target sequence (e.g., agenomic safe harbor site). Additionally, the CRISPR-Cas nuclease candirect cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from thefirst or last nucleotide of a target sequence.

The presently disclosed nucleases and CRISPR-Cas system allow fortargeted delivery of the expression cassette. In certain embodiments, apresently disclosed CRISPR-Cas system or the DNA binding domain of apresently disclosed nuclease binds to a genomic safe harbor site. Anuclease or the CRISPR-Cas system generates a double strand break at thegenomic safe harbor site. Genomic safe harbor sites are intragenic orextragenic regions of the human genome that are able to accommodate thepredictable expression of newly integrated DNA without adverse effectson the host cell or organism. A useful safe harbor must permitsufficient transgene expression to yield desired levels of thevector-encoded protein or non-coding RNA. A genomic safe harbor sitealso must not predispose cells to malignant transformation nor altercellular functions. Methods for identifying genomic safe harbor sitesare described in Sadelain et al., “Safe Harbours for the integration ofnew DNA in the human genome,” Nature Reviews (2012); 12:51-58;Papapetrou et al., “Genomic safe harbors permit high β-globin transgeneexpression in thalassemia induced pluripotent stem cells” NatBiotechnol. (2011) January; 29(1):73-8, which are incorporated byreference in their entireties. A presently disclosed genomic safe harborsite meets one or more (one, two, three, four, or five) of the followingfive criteria: (1) distance of at least 50 kb from the 5′ end of anygene (e.g., from the 5′ end of the gene), (ii) distance of at least 300kb from any cancer-related gene, (iii) within an open/accessiblechromatin structure (measured by DNA cleavage with natural or engineerednucleases), (iv) location outside a gene transcription unit and (v)location outside ultraconserved regions (UCRs), microRNA or longnon-coding RNA of the human genome. As the most common insertionaloncogenesis event is transactivation of neighboring tumor-promotinggenes, the first two criteria exclude the portion of the human genomelocated near promoters of genes, in particular, cancer-related genes,which are genes functionally implicated in human cancers or the humanhomologs of genes implicated in cancer in model organisms. Proximity tomiRNA genes is one exclusion criterion because miRNAs are implicated inthe regulation of many cellular processes, including cell proliferationand differentiation. As vector integration within a transcription unitcan disrupt gene function through the loss of function of a tumorsuppressor gene or the generation of an aberrantly spliced gene product,the fourth (iv) criterion excludes all sites located inside transcribedgenes. UCRs, which are regions that are highly conserved over multiplevertebrates and known to be enriched for enhancers and exons, and longnon-coding RNAs, are also excluded. In certain embodiments, the genomicsafe harbor site is an extragenic genomic safe harbor site. In certainembodiments, the genomic safe harbor site is located on chromosome 1.

The presently disclosed subject matter also provides polynucleotidesencoding the above-described nucleases, vectors comprising thepolynucleotides encoding the above-described nucleases, polynucleotidesencoding the above-described CRISPR-Cas system, and vectors comprisingthe polynucleotides encoding the above-described CRISPR-Cas system.

The nucleases and polynucleotides encoding these nucleases, and theCRISPR-Cas system and polynucleotides encoding the CRISPR-Cas system canbe delivered in vivo or ex vivo by any suitable means. For example,nucleases and CRISPR-Cas system as described herein can be delivered toa cell (e.g., a hematopoietic stem cell, an embryonic stem cell, aninduced pluripotent stem cell, or an hemogenic endothelium cell) by avector comprising polynucleotides encoding the nuclease or theCRISPR-Cas system. Any vectors can be used including, but not limitedto, plasmid vectors, retroviral vectors (e.g., γ-retroviral vectors,lentiviral vectors and foamy viral vectors), adenovirus vectors,poxvirus vectors; herpes virus vectors and adena-associated virusvectors, etc. In one embodiment, the vector comprising a polynucleotideencoding an above-described nuclease or an above-described CRISPR-Cassystem is a lentiviral vector. In one particular embodiment, thelentiviral vector is a non-integrating lentiviral vector. Examples ofnon-integrating lentiviral vector are described in Ory et al. (1996)Proc. Natl. A cad. Sci. USA 93:11382-11388; Dull et al., (1998) J.Viral. 72:8463-8471; Zuffery et al. (1998) J. Viral. 72:9873-9880;Follenzi et al., (2000) Nature Genetics 25:217-222; U.S. PatentPublication No 2009/054985.

Additionally, non-viral approaches can also be employed for theexpression of a globin gene in cells. For example, a nucleic acidmolecule can be introduced into a cell by administering the nucleic acidin the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci.U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990;Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al.,Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysineconjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988;Wu et al., Journal of Biological Chemistry 264:16985, 1989), or bymicro-injection under surgical conditions (Wolff et al., Science247:1465, 1990). Other non-viral means for gene transfer includetransfection in vitro using calcium phosphate, DEAE dextran,electroporation, and protoplast fusion. Liposomes can also bepotentially beneficial for delivery of DNA into a cell. Transplantationof normal genes into the affected tissues of a subject can also beaccomplished by transferring a normal nucleic acid into a cultivatablecell type ex vivo (e.g., an autologous or heterologous primary cell orprogeny thereof), after which the cell (or its descendants) are injectedinto a targeted tissue or are injected systemically. Recombinantreceptors can also be derived or obtained using transposases. Transientexpression may be obtained by RNA electroporation.

IV. CELLS

Genetic modification of cells (e.g., hematopoietic stem cells, embryonicstem cells, induced pluripotent stem cells, and hemogenic endotheliumcells) can be accomplished by transducing a substantially homogeneouscell composition with a recombinant DNA or RNA construct (e.g., a vectoror a delivery system comprising the above-described expressioncassette). The presently disclosed subject matter provides cellstransduced with the above-described expression cassettes, cellstransduced with the above-described vectors, and cells transduced withthe above-described nucleases or with vectors comprising polynucleotidesencoding the nucleases, and cell transduced with the above-describedCARISPR-Cas system or with vectors comprising polynucleotides encodingthe CARISPR-Cas system, which are collectively referred to as“transduced cells”. As described above, the vectors, nucleases andCRISPR-Cas system are employed for transduction of the expressioncassette to the cells to express a globin gene (e.g., a human β-globingene). In certain embodiments, the transduced cells are administered toa subject to treat and/or prevent a hematopoietic disease, disorder, orcondition. The presently disclosed insulators can enhance the efficiencyof the transduction of the expression cassette to cells.

Suitable transduced cells include, but are not limited to, stem cells,progenitor cells, and differentiated cells. As used herein, the term“progenitor” or “progenitor cells” refers to cells that have thecapacity to self-renew and to differentiate into more mature cells.Progenitor cells have a reduced potency compared to pluripotent andmultipotent stem cells. Many progenitor cells differentiate along asingle lineage, but may also have quite extensive proliferativecapacity.

In certain embodiments, the transduced cells are stem cells. Stem cellshave the ability to differentiate into the appropriate cell types whenadministered to a particular biological niche, in vivo. A stem cell isan undifferentiated cell capable of (1) long term self-renewal, or theability to generate at least one identical copy of the original cell,(2) differentiation at the single cell level into multiple, and in someinstance only one, specialized cell type and (3) of in vivo functionalregeneration of tissues. Stem cells are sub-classified according totheir developmental potential as totipotent, pluripotent, multipotentand oligo/unipotent. As used herein, the term “pluripotent” means theability of a cell to form all lineages of the body or soma (i.e., theembryo proper). For example, embryonic stem cells are a type ofpluripotent stem cells that are able to form cells from each of thethree germs layers, the ectoderm, the mesoderm, and the endoderm. Asused herein, the term “multipotent” refers to the ability of an adultstem cell to form multiple cell types of one lineage. For example,hematopoietic stem cells are capable of forming all cells of the bloodcell lineage, e.g., lymphoid and myeloid cells.

In certain embodiments, the transduced cells are embryonic stem cells,bone marrow stem cells, umbilical cord stem cells, placental stem cells,mesenchymal stem cells, neural stem cells, liver stem cells, pancreaticstem cells, cardiac stem cells, kidney stem cells, and/or hematopoieticstem cells. In one embodiment, the transduced cells are hematopoieticstem cells (HSCs). HSCs give rise to committed hematopoietic progenitorcells (HPCs) that are capable of generating the entire repertoire ofmature blood cells over the lifetime of an organism. The term“hematopoietic stem cell” or “HSC” refers to multipotent stem cells thatgive rise to all blood cell types of an organism, including myeloid(e.g., monocytes and macrophages, neutrophils, basophils, eosinophils,erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoidlineages (e.g., T-cells, B-cells, NK-cells). When transplanted intolethally irradiated animals or humans, hematopoietic stem and progenitorcells can repopulate the erythroid, neutrophil-macrophage, megakaryocyteand lymphoid hematopoietic cell pool.

HSCs can be isolated or collected from bone marrow, umbilical cordblood, or peripheral blood. HSCs can be identified according to certainphenotypic or genotypic markers. For example, HSCs can be identified bytheir small size, lack of lineage (lin) markers, low staining (sidepopulation) with vital dyes such as rhodamine 123 (rhodamineDULL, alsocalled rholo) or Hoechst 33342, and presence of various antigenicmarkers on their surface, many of which belong to the cluster ofdifferentiation series (e.g., CD34, CD38, CD90, CD133, CD105, CD45, Terl19, and c-kit, the receptor for stem cell factor). In one embodiment,the transduced cell is a CD34⁺ HSC.

In one embodiment, the transduced cell is an embryonic stem cell. Inanother embodiment, the transduced cell is an induced pluripotent stemcell. In yet another embodiment, the transduced cell is a hemogenicendothelium cell.

While HSCs are the natural vehicle for restoring long-termhematopoiesis, their use has some important limitations. The first istheir relative scarcity, which can eventually preclude autologous HSCtherapy when the harvested cellular product is too small. The second isthe difficulty to perform biosafety testing such as integration siteanalysis and consequently to select cells with chosen integration sites,because adult HSCs cannot be replicated in vitro. The third limitationis that homologous recombination using current technologies ispractically impossible thus compromising the advent of gene correction.All of these limitations are ultimately due to the fact that adult HSCscannot be expanded in vitro without losing their stem cell potency.These limitations explain the critical importance of viral vectors suchas gamma-retroviral and lentiviral vectors, which are remarkably quickand efficient in achieving stable gene transfer. This is essential whendealing with HSCs that are only available in limited quantities.

Use of ESs and induced pluripotent stem (iPS) cells for globin genetherapy is disclosed in Moi et al., Haematol Mar. 1, 2008;93(3):325-330. Embryonic stem (ES) cells are amenable to gene targetingand correction, which requires unlimited in vitro cell division withoutlosing multipotency. Chang et al., Proc Natl Acad Sci USA 2006;103:1036-40 provided proof of principle of the feasibility of thehomologous recombination approach in mice with sickle cell anemia.Takahashi et al. Cell 2006; 126:663-76 reported the successfulreprogramming of fibroblasts to an embryonic stem-like state. Cellsobtained by this reverse-differentiation process, called inducedpluripotent stem (iPS) cells, were produced by exposing embryonic oryoung adult bulk fibroblast cultures to gamma-retroviral vectorsencoding 4 transcription factors, which are physiologically active inthe embryonic stem cells, but generally turned off when differentiationprogresses. The cultured cells formed colonies similar to ES cellcolonies. These findings have been confirmed and extended by others toboth mouse and human fibroblasts (Meissner et al., Nat Biotechnol 2007;25:1177-81; Nakagawa et al., Nat Biotechnol 2007; 26:101-6; Okita etal., Nature 2007; 448:313-7; Park et al., Nature 2007; 451:141-6;Takahashi et al., Nat Protoc 2007; 2:3081-9; Takahashi K et al., Cell2007; 131:861-72; Wernig et al., Nature 2007; 448:318-24; Yu J et al.,Science 2007; 318: 1917-20). Rudolf Jaenisch and co-workers achieved asuccessful gene therapy in a mouse model of sickle cell disease, usinghomologous recombination in ES-like iPS cells (Hanna et al., Science2007; 318:1920-3). The process has so far been mostly applied tofibroblast harvested from a skin biopsy, which are then induced tobecome iPS by transduction with retroviral vectors that encode four stemcell transcription factors. iPS are amenable to the correction of the SCmutation by standard homologous recombination techniques and can then bedifferentiated in vitro into unlimited amounts of hematopoietic stemcells. The whole process ends with the autologous transplantation of thecorrected HSC into the original mouse donor, which will now be cured ofits SC disease. This technique is not only useful for homologousrecombination, but can also enhance lentiviral-mediated globin genetransfer for the treatment of β-thalassemia by providing a means toperform detailed integration site analysis and adequate in vitro cellexpansion before infusing cells into the recipient.

The cell of the presently disclosed subject matter can be autologous(“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic orxenogeneic). As used herein, “autologous” refers to cells from the samesubject. As used herein, “allogeneic” refers to cells of the samespecies that differ genetically to the cell in comparison. As usedherein, “syngeneic” refers to cells of a different subject that aregenetically identical to the cell in comparison. As used herein,“xenogeneic” refers to cells of a different species to the cell incomparison. In certain embodiments, the cell is autologous, e.g., a celltransduced with the presently disclosed expression cassette isadministered to a subject from whom the cell is collected, e.g., thecell is collected from bone marrow, umbilical cord blood, peripheralblood, and/or adipose tissue of the subject. In certain embodiments, thecell is obtained or collected from bone marrow of a subject.

In certain embodiments, prior to transduction with the expressioncassette, the cell is pre-stimulated, e.g., in the presence of one ormore cytokines (e.g., IL-3, IL-1α, IL-6, Kit ligand (also known as “StemCell Factor (SCF)”), and Flt-3 ligand), and/or one or more glycoproteins(e.g., thrombopoietin and fibronectin). In one non-limiting example, thecell is pre-stimulated in the presence of Flt-3 ligand, SCF,thrombopoietin, interleukin-3, and fibronectin. The cell can bepre-stimulated for about 24 hours or longer, e.g., about 48 hours, orabout 36 hours. Subsequently, the cell is transduced with a presentlydisclosed expression cassette, or a vector or another delivery systemcomprising such expression cassette. Transduction can be performed on afresh cell, or on a frozen cell. Genomic DNA of the cell is isolated todetermine the vector copy number and analyze the integration site orintegrated vector structure, e.g., by South blot analysis and/or byQuantitative PCR. For quantification of globin mRNA (e.g., humanβ-globin transgene analysis), total RNA is extracted from the cell.Quantitative primer extension assay can be used for quantification ofglobin mRNA.

V. COMPOSITIONS AND FORMULATIONS

The presently disclosed subject matter provides pharmaceuticalcompositions comprising a presently disclosed transduced cell asdescribed above and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible, including pharmaceutically acceptable cellculture media. The pharmaceutically acceptable carrier can be suitablefor parenteral (e.g., intravenous, intramuscular, subcutaneous, orintraperitoneal), spinal or epidermal administration (e.g., byinjection, infusion or implantation). Depending on the route ofadministration, the active compound, e.g., the transduced cell, may becoated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe transduced cells, use thereof in the pharmaceutical compositions ofthe invention is contemplated.

The pharmaceutical compositions of the presently disclosed subjectmatter can further comprise one or more polypeptides, polynucleotides,vectors comprising the same, transduced cells, etc., as describedherein, formulated in pharmaceutically-acceptable orphysiologically-acceptable solutions for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy. If desired, the pharmaceutical compositions ofthe presently disclosed subject matter can be administered incombination with other agents, including, but not limited to, cytokines,growth factors, hormones, small molecules or variouspharmaceutically-active agents. Any additional agents that do notadversely affect the ability of the composition to deliver the intendedgene therapy can be included in the compositions.

In the pharmaceutical compositions of the presently disclosed subjectmatter, formulation of pharmaceutically-acceptable excipients andcarrier solutions is well known to those of ordinary skill in the art,as is the development of suitable dosing and treatment regimens forusing the particular compositions described herein in a variety oftreatment regimens, including, e.g., oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation.

The pharmaceutical compositions of the presently disclosed subjectmatter can be delivered parenterally (e.g., intravenously,intramuscularly, or intraperitoneally) as described, for example, inU.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363. Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The pharmaceutically acceptablecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The pharmaceutical compositions of the presently disclosed subjectmatter can be conveniently provided as sterile liquid preparations,e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions,or viscous compositions, which can be buffered to a selected pH. Liquidpreparations are normally easier to prepare than gels, other viscouscompositions, and solid compositions. Additionally, liquid compositionsare somewhat more convenient to administer, especially by injection.Viscous compositions, on the other hand, can be formulated within theappropriate viscosity range to provide longer contact periods withspecific tissues. Liquid or viscous compositions can comprise carriers,which can be a solvent or dispersing medium containing, for example,water, saline, phosphate buffered saline, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like) and suitablemixtures thereof.

Sterile injectable solutions can be prepared by incorporating thecompositions of the presently disclosed subject matter in the requiredamount of the appropriate solvent with various amounts of the otheringredients, as desired. Such compositions may be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, dextrose, or the like. The compositionscan also be lyophilized. The compositions can contain auxiliarysubstances such as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like,depending upon the route of administration and the preparation desired.Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17thedition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations, without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added.

Prevention of the action of microorganisms can be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorptionof the injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, alum inurn monostearate andgelatin.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of the presently disclosed subject matter can beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Sterileinjectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

In certain embodiments, the compositions can be delivered by intranasalsprays, inhalation, and/or other aerosol delivery vehicles. Methods fordelivering genes, polynucleotides, and peptide compositions directly tothe lungs via nasal aerosol sprays are described, e.g., in U.S. Pat. No.5,756,353 and U.S. Pat. No. 5,804,212. Methods of delivering drugs usinglysophosphatidyl-glycerol compounds are described, e.g., in U.S. Pat.No. 5,725,871. Transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described, e.g., in U.S. Pat.No. 5,780,045. The compositions of the presently disclosed subjectmatter can be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, a nanoparticle or thelike. The formulation and use of such delivery vehicles can be carriedout using known and conventional techniques. The formulations andcompositions of the presently disclosed subject matter can comprise oneor more repressors and/or activators comprising a combination of anynumber of polypeptides, polynucleotides, and small molecules, asdescribed herein, formulated in pharmaceutically-acceptable orphysiologically-acceptable solutions (e.g., culture medium) foradministration to a cell or an animal, either alone, or in combinationwith one or more other modalities of therapy.

In certain aspects, the presently disclosed subject matter providesformulations or compositions suitable for the delivery of viral vectorsystems (i.e., viral-mediated transduction) including, but not limitedto, retroviral (e.g., lentiviral) vectors. Exemplary formulations for exvivo delivery can also include the use of various transfection agentsknown in the art, such as calcium phosphate, electoporation, heat shockand various liposome formulations (i.e., lipid-mediated transfection).Liposomes are lipid bilayers entrapping a fraction of aqueous fluid. DNAspontaneously associates to the external surface of cationic liposomes(by virtue of its charge) and these liposomes will interact with thecell membrane.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the presently disclosed subject matter.Typically, any additives (in addition to the transduced cell(s) and/oragent(s)) are present in an amount of from about 0.001% to about 50% byweight) solution in phosphate buffered saline, and the active ingredientis present in the order of micrograms to milligrams, such as from about0.0001 wt % to about 5 wt %, from about 0.0001 wt % to about 1 wt %,from about 0.0001 wt % to about 0.05 wt %, from about 0.001 wt % toabout 20 wt %, from about 0.01 wt % to about 10 wt %, or from about 0.05wt % to about 5 wt %. For any composition to be administered to ananimal or human, and for any particular method of administration,toxicity should be determined, such as by determining the lethal dose(LD) and LD50 in a suitable animal model e.g., rodent such as mouse;and, the dosage of the composition(s), concentration of componentstherein and timing of administering the composition(s), which elicit asuitable response. Such determinations do not require undueexperimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the time for sequentialadministrations can be ascertained without undue experimentation.

VI. USES AND METHODS

Vectors and other delivery systems (nucleases and CRISPR-Cas systems)comprising the presently disclosed expression cassette provide improvedmethods of gene therapy. As used herein, the term “gene therapy” refersto the introduction of a polynucleotide into a cell's genome thatrestores, corrects, or modifies the gene and/or expression of the gene.In various non-limiting embodiments, a presently disclosed vector orother delivery system (e.g., a nuclease or a CRISPR-Cas system)comprises an expression cassette comprising a globin gene or afunctional portion thereof that encodes a globin protein (e.g., human βglobin protein), which provides curative, preventative, or ameliorativebenefits to a subject diagnosed with or that is suspected of having adisease, disorder, or condition of the hematopoietic system. The vectoror other delivery systems (e.g., a nuclease and the CRISPR-Cas system)can infect and transduce the cell in vivo, ex vivo, or in vitro. In exvivo and in vitro embodiments, the transduced cells can then beadministered to a subject in need of therapy. The presently disclosedsubject matter contemplates that the vectors and other delivery systems(e.g., nucleases or CRISPR-Cas systems), viral particles, and transducedcells of the presently disclosed subject matter are be used to treat,prevent, and/or ameliorate a disease, disorder, or condition of thehematopoietic system in a subject, e.g., a hemoglobinopathy.

As used herein, the term “hemoglobinopathy” or “hemoglobinopathiccondition” includes any disorder involving the presence of an abnormalhemoglobin molecule in the blood. Examples of hemoglobinopathiesincluded, but are not limited to, hemoglobin C disease, hemoglobinsickle cell disease (SCD), sickle cell anemia, and thalassemias. Alsoincluded are hemoglobinopathies in which a combination of abnormalhemoglobins are present in the blood (e.g., sickle cell/Hb-C disease).

As used herein, “thalassemia” refers to a hereditary disordercharacterized by defective production of hemoglobin. Examples ofthalassemias include α- and β-thalassemia. β-thalassemias are caused bya mutation in the beta globin chain, and can occur in a major or minorform. In the major form of β-thalassemia, children are normal at birth,but develop anemia during the first year of life. The mild form ofβ-thalassemia produces small red blood cells and the thalassemias arecaused by deletion of a gene or genes from the globin chain.α-thalassemia typically results from deletions involving the HBA1 andHBA2 genes. Both of these genes encode α-globin, which is a component(subunit) of hemoglobin. There are two copies of the HBA1 gene and twocopies of the HBA2 gene in each cellular genome. As a result, there arefour alleles that produce α-globin. The different types of a thalassemiaresult from the loss of some or all of these alleles. Hb Bart syndrome,the most severe form of a thalassemia, results from the loss of all fourα-globin alleles. HbH disease is caused by a loss of three of the four[alpha]-globin alleles. In these two conditions, a shortage of[alpha]-globin prevents cells from making normal hemoglobin. Instead,cells produce abnormal forms of hemoglobin called hemoglobin Bart (HbBart) or hemoglobin H (HbH). These abnormal hemoglobin molecules cannoteffectively carry oxygen to the body's tissues. The substitution of HbBart or HbH for normal hemoglobin causes anemia and the other serioushealth problems associated with a thalassemia.

As used herein, the term “sickle cell disease” refers to a group ofautosomal recessive genetic blood disorders, which results frommutations in a globin gene and which is characterized by red blood cellsthat assume an abnormal, rigid, sickle shape. They are defined by thepresence of β^(S)-gene coding for a β-globin chain variant in whichglutamic acid is substituted by valine at amino acid position 6 of thepeptide, and second β-gene that has a mutation that allows for thecrystallization of HbS leading to a clinical phenotype. As used herein,the term “sickle cell anemia” refers to a specific form of sickle celldisease in patients who are homozygous for the mutation that causes HbS.Other common forms of sickle cell disease include HbS/β-thalassemia,HbS/HbC and HbS/HbD.

In certain embodiments, gene therapy methods of the presently disclosedsubject mater are used to treat, prevent, or ameliorate ahemoglobinopathy that is selected from the group consisting of:hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cellanemia, hereditary anemia, thalassemia, β-thalassemia, thalassemiamajor, thalassemia intermedia, α-thalassemia, and hemoglobin H disease.In one non-limiting embodiment, the hemoglobinopathy is β-thalassemia.In another non-limiting embodiment, the hemoglobinopathy is sickle cellanemia

In various non-limiting embodiments, vectors or other delivery systems(e.g., nucleases or CRISPR-Cas systems) comprising a presently disclosedexpression cassette are administered by direct injection to a cell,tissue, or organ of a subject in need of gene therapy, in vivo. Invarious other embodiments, cells are transduced in vitro or ex vivo withvectors or other delivery systems (e.g., nucleases or CRISPR-Cassystems) of the presently disclosed subject matter, and optionallyexpanded ex vivo. The transduced cells are then administered to asubject in need of gene therapy, e.g., within a pharmaceuticalformulation disclosed herein.

The presently disclosed subject matter provides a method of providing atransduced cell to a subject. In various non-limiting embodiments, themethod comprises administering (e.g., parenterally) one or more cells (apopulation of cells) transduced with a presently disclosed expressioncassette or a vector or another delivery system (e.g., a nuclease orCRISPR-Cas system) comprising such expression cassette to the subject.

The presently disclosed subject matter provides a method of treating ahemoglobinopathy in a subject. In various non-limiting embodiments, themethod comprises administering an effective amount of a presentlydisclosed transduced cell or a population of the presently disclosedtransduced cells (e.g., HSCs, embryonic stem cells, or iPSCs) to thesubject.

For treatment, the amount administered is an amount effective inproducing the desired effect. An effective amount can be provided in oneor a series of administrations. An effective amount can be provided in abolus or by continuous perfusion. An “effective amount” (or“therapeutically effective amount”) is an amount sufficient to affect abeneficial or desired clinical result upon treatment. An effectiveamount can be administered to a subject in one or more doses. In termsof treatment, an effective amount is an amount that is sufficient topalliate, ameliorate, stabilize, reverse or slow the progression of thedisease, or otherwise reduce the pathological consequences of thedisease. The effective amount is generally determined by the physicianon a case-by-case basis and is within the skill of one in the art.Several factors are typically taken into account when determining anappropriate dosage to achieve an effective amount. These factors includeage, sex and weight of the subject, the condition being treated, theseverity of the condition and the form and effective concentration ofthe immunoresponsive cells administered.

In one non-limiting example, following administration of one or more ofthe presently disclosed transduced cells, peripheral blood of thesubject is collected and hemoglobin levels is measured. Atherapeutically relevant level of hemoglobin is produced followingadministration of one or more of the presently disclosed transducedcells. Therapeutically relevant level of hemoglobin is a level ofhemoglobin that is sufficient (1) to improve or correct anemia, (2) torestore the ability of the subject to produce red blood cells containingnormal hemoglobin, (3) to correct ineffective erythropoiesis in thesubject, (4) to correct extra-medullary hematopoiesis (e.g., splenic andhepatic extra-medullary hematopoiesis), and/or (5) to reduce ironaccumulation, e.g., in peripheral tissues and organs. Therapeuticallyrelevant level of hemoglobin can be at least about 7 g/dL Hb, at leastabout 7.5 g/dL Hb, at least about 8 g/dL Hb, at least about 8.5 g/dL Hb,at least about 9 g/dL Hb, at least about 9.5 g/dL Hb, at least about 10g/dL Hb, at least about 10.5 g/dL Hb, at least about 11 g/dL Hb, atleast about 11.5 g/dL Hb, at least about 12 g/dL Hb, at least about 12.5g/dL Hb, at least about 13 g/dL Hb, at least about 13.5 g/dL Hb, atleast about 14 g/dL Hb, at least about 14.5 g/dL Hb, or at least about15 g/dL Hb. Additionally or alternatively, therapeutically relevantlevel of hemoglobin can be from about 7 g/dL Hb to about 7.5 g/dL Hb,from about 7.5 g/dL Hb to about 8 g/dL Hb, from about 8 g/dL Hb to about8.5 g/dL Hb, from about 8.5 g/dL Hb to about 9 g/dL Hb, from about 9g/dL Hb to about 9.5 g/dL Hb, from about 9.5 g/dL Hb to about 10 g/dLHb, from about 10 g/dL Hb to about 10.5 g/dL Hb, from about 10.5 g/dL Hbto about 11 g/dL Hb, from about 11 g/dL Hb to about 11.5 g/dL Hb, fromabout 11.5 g/dL Hb to about 12 g/dL Hb, from about 12 g/dL Hb to about12.5 g/dL Hb, from about 12.5 g/dL Hb to about 13 g/dL Hb, from about 13g/dL Hb to about 13.5 g/dL Hb, from about 13.5 g/dL Hb to about 14 g/dLHb, from about 14 g/dL Hb to about 14.5 g/dL Hb, from about 14.5 g/dL Hbto about 15 g/dL Hb, from about 7 g/dL Hb to about 8 g/dL Hb, from about8 g/dL Hb to about 9 g/dL Hb, from about 9 g/dL Hb to about 10 g/dL Hb,from about 10 g/dL Hb to about 11 g/dL Hb, from about 11 g/dL Hb toabout 12 g/dL Hb, from about 12 g/dL Hb to about 13 g/dL Hb, from about13 g/dL Hb to about 14 g/dL Hb, from about 14 g/dL Hb to about 15 g/dLHb, from about 7 g/dL Hb to about 9 g/dL Hb, from about 9 g/dL Hb toabout 11 g/dL Hb, from about 11 g/dL Hb to about 13 g/dL Hb, or fromabout 13 g/dL Hb to about 15 g/dL Hb. In certain embodiments, thetherapeutically relevant level of hemoglobin is maintained in thesubject for at least about 6 months, for at least about 12 months (or 1year), for at least about 24 months (or 2 years). In certainembodiments, the therapeutically relevant level of hemoglobin ismaintained in the subject for up to about 6 months, for up to about 12months (or 1 year), for up to about 24 months (or 2 years). In certainembodiments, the therapeutically relevant level of hemoglobin ismaintained in the subject for about 6 months, for about 12 months (or 1year), for about 24 months (or 2 years). In certain embodiments, thetherapeutically relevant level of hemoglobin is maintained in thesubject for from about 6 months to about 12 months (e.g., from about 6months to about 8 months, from about 8 months to about 10 months, fromabout 10 months to about 12 months), from about 12 months to about 18months (e.g., from about 12 months to about 14 months, from about 14months to about 16 months, or from about 16 months to about 18 months),or from about 18 months to about 24 months (e.g., from about 18 monthsto about 20 months, from about 20 months to about 22 months, or fromabout 22 months to about 24 months).

In certain embodiments, the method comprises administering one or morecells transduced with a recombinant vector comprising a presentlydisclosed expression cassette as described above. The vector copy numberof the recombinant vector in the cells that provide for thetherapeutically relevant level of hemoglobin (e.g., 9-10 g/dL) in thesubject is from about 0.5 to about 2, from about 0.5 to about 1, or fromabout 1 to about 2 vector copy number per cell. In certain embodiments,the vector copy number of the presently disclosed vector is about 0.5,about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8,about 1.9, or about 2.0 vector copy number per cell.

In certain embodiments, the subject lacks a human leukocyte antigen(HLA)-matched donor. In certain embodiments, the transduced cell is fromthe same subject. In one embodiment, the transduced cell is from bonemarrow of the same subject. Thus, administration of the transduced cellsdo not incur the risk of graft-versus host disease in the subject. Themethod does not require immune suppression to prevent graft rejection,e.g., the method does not comprise administering an immunosuppressiveagent to the subject.

The present disclosed subject matter also provides a method ofincreasing the proportion of red blood cells or erythrocytes compared towhite blood cells or leukocytes in a subject. In various non-limitingembodiments, the method comprises administering an effective amount of apresently disclosed transduced cell or a population of the presentlydisclosed transduced cells (e.g., HSCs, embryonic stem cells, or iPSCs)to the subject, wherein the proportion of red blood cell progeny cellsof the hematopoietic stem cells are increased compared to white bloodcell progeny cells of the hematopoietic stem cells in the subject.

Without wishing to be bound to any particular theory, an importantadvantage provided by the expression cassette, vectors and otherdelivery systems (e.g., nucleases and CRISPR-Cas systems), compositions,and methods of the presently disclosed subject is the high efficacy ofglobin gene therapy that can be achieved by administering populations ofcells comprising lower percentages of transduced cells compared toexisting methods. This provides important safety advantages associatedwith reduced chances of deleterious mutation, transformation, oroncogene activation of cellular genes in transduced cells. Thetransduced cells can be administered as part of a bone marrow or cordblood transplant in an individual that has or has not undergone bonemarrow ablative therapy.

One consideration concerning the therapeutic use of the presentlydisclosed cells transduced with the expression cassette described herein(“transduced cells”) is the quantity of cells necessary to achieve anoptimal effect. The quantity of transduced cells to be administered willvary for the subject being treated. In one embodiment, from about 1×10⁴to about 1×10⁵ cells/kg, from about 1×10⁵ to about 1×10⁶ cells/kg, fromabout 1×10⁶ to about 1×10⁷ cells/kg, from about 1×10⁷ to about 1×10⁸cells/kg, from about 1×10⁸ to about 1×10⁹ cells/kg, or from about 1×10⁹to about 1×10¹⁰ cells/kg of the presently disclosed transduced cells areadministered to a subject. More effective cells may be administered ineven smaller numbers. In some embodiments, at least about 1×10⁸cells/kg, at least about 2×10⁸ cells/kg, at least about 3×10⁸ cells/kg,at least about 4×10⁸ cells/kg, or at least about 5×10⁸ cells/kg of thepresently disclosed transduced cells are administered to a subject. Theprecise determination of what would be considered an effective dose maybe based on factors individual to each subject, including their size,age, sex, weight, and condition of the particular subject. Dosages canbe readily ascertained by those skilled in the art from this disclosureand the knowledge in the art.

In various embodiments, the expression cassettes, vectors and otherdelivery systems (nucleases and CRISPR-Cas systems), compositions, andmethods of the presently disclosed subject matter offer improved methodsof gene therapy using ex vivo gene therapy and autologoustransplantation. Transplantation of cells transduced with the expressioncassette or into subjects having a hemoglobinopathy results in long-termcorrection of the disease.

One or more presently disclosed transduced cells can be administered byany methods known in the art, including, but not limited to, parenteraladministration (e.g., intramuscular administration, intravenousadministration, subcutaneous administration, or intraperitonealadministration), spinal administration, and epidermal administration. Inone non-limiting embodiment, one or more transduced cells are deliveredto a subject intravenously. One or more presently disclosed transducedcells can be administered by injection, infusion, or implantation. Inone non-limiting embodiment, one or more transduced cells areadministered by injection. In another non-limiting embodiment, one ormore transduced cells are administered by intravenous injection.

The subjects can have an advanced form of disease, in which case thetreatment objective can include mitigation or reversal of diseaseprogression, and/or amelioration of side effects. The subjects can havea history of the condition, for which they have already been treated, inwhich case the therapeutic objective will typically include a decreaseor delay in the risk of recurrence.

VII. KITS

The presently disclosed subject matter provides kits for the treatmentor prevention of a hemoglobinopathy. In one embodiment, the kitcomprises a therapeutic or prophylactic composition containing aneffective amount of a cell transduced with the presently disclosedexpression cassette in unit dosage form. In one non-limiting embodiment,the kit comprises one or more expression cassettes disclosed herein. Incertain embodiments, the kit comprises one or more vectors comprising anexpression cassette disclosed herein. In some embodiments, the kitcomprises a sterile container, which can be a box, an ampule, a bottle,a vial, a tube, a bag, a pouch, a blister-pack, or other suitablecontainer forms known in the art. Such containers can be made ofplastic, glass, laminated paper, metal foil, or other materials suitablefor holding medicaments.

If desired, the transduced cell is provided together with instructionsfor administering the cell to a subject having or at risk of developinga hemoglobinopathy. The instructions will generally include informationabout the use of the composition for the treatment or prevention of ahemoglobinopathy. In other embodiments, the instructions include atleast one of the following: description of the therapeutic agent; dosageschedule and administration for treatment or prevention of ahemoglobinopathy or symptoms thereof; precautions; warnings;indications; counter-indications; overdosage information; adversereactions; animal pharmacology; clinical studies; and/or references.Alternatively or additionally, the kit can include instructions fortransducing a cell with the one or more expression cassettes and/orvectors comprising such expression cassettes. The instructions may beprinted directly on the container (when present), or as a label appliedto the container, or as a separate sheet, pamphlet, card, or foldersupplied in or with the container.

EXAMPLES

The practice of the presently disclosed subject matter employs, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are well within the purview of theskilled artisan. Such techniques are explained fully in the literature,such as, “Molecular Cloning: A Laboratory Manual”, second edition(Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal CellCulture” (Freshney, 1987); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1996); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Current Protocols inMolecular Biology” (Ausubel, 1987); “PCR: The Polymerase ChainReaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan,1991). These techniques are applicable to the production of thepolynucleotides and polypeptides of the presently disclosed subjectmatter, and, as such, may be considered in making and practicing thepresently disclosed subject matter. Particularly useful techniques forparticular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the expression cassettes, vectors, delivery systems, andtherapeutic methods of the presently disclosed subject matter, and arenot intended to limit the scope of what the inventors regard as theirinvention.

Example 1: Discovery of Novel Insulators

The problems created by insertional mutagenesis of viral vectors arewidely known (Nienhuis (2013), Baum et al. (2006), Nienhuis et al.(2006)) as is the evidence that the risks of genotoxicity can be reducedby the use of chromatin insulators (Arumugam et al. (2007), Emery(2011), Evans-Galea et al. (2007), Rivella et al. (2000), Emery et al.(2000), Emery et al. (2002), Yannaki et al. (2002), Hino et al. (2004),Ramezani et al. (2003), Ramezani et al. (2008)). Approaches allowing theefficient identification of enhancer blocking insulators in the humangenome have been developed. These new insulators are short, on theaverage 150 bp, and they do not affect adversely the titers of viralvectors and they are several times more powerful than the insulatorcHS4. Genomic approaches were used to discover the most powerfulenhancer blocker and barrier insulators of the human genome. For genetherapy of the hemoglobinopathies, powerful enhancers are required toachieve therapeutic levels of globin gene expression. Powerfulinsulators may thus provide one means to protect the genomic environmentfrom the powerful enhancers of the integrating vectors.

Several studies have demonstrated the ability of the cHS4 insulator toreduce position-effect silencing of gammaretroviral vectors (Evans-Galeaet al. (2007), Rivella et al. (2000), Emery et al. (2000), Emery et al.(2002), Yannaki et al. (2002), Hino et al. (2004), Ramezani et al.(2006), Yao et al. (2003), Nishino et al. (2006), Aker et al. (2007), Liand Emery (2008)), and lentiviral vectors (Evans-Galea et al. (2007),Ramezani et al. (2003), Puthenveetil et al. (2004), Arumugam et al.(2007), Bank et al. (2005), Aker et al. (2007), Ma et al. (2003), Changet al. (2005), Pluta et al. (2005)). Those studies that wereappropriately designed demonstrated that inclusion of the 1.2 kb versionof the cHS4 insulator increased the likelihood and/or consistency ofvector transgene expression in at least some settings (Arumugam et al.(2007), Evans-Galea et al. (2007), Emery et al. (2002), Yannaki et al.(2002), Hino et al. (2004), Ramezani et al. (2006), Aker et al. (2007),Li and Emery (2008), Pluta et al. (2005), Jakobsson et al. (2004)).Nevertheless, the degree of protection afforded by the cHS4 insulator isfar from complete. In addition, the inclusion of the 1.2 Kb cHS4 canadversely affect vector titers while the smallest cHS4 core has beenproven ineffective (Aker et al. (2007), Jakobsson et al. (2004)).

Effects on genotoxicity were tested using an in vivo assay based onquantitation of tumor formation in mice. Vectors insulated by insulatorA1 decreased tumor formation induced by random vector integration inhematopoietic chimeras compared to mice that received uninsulated orcHS4-insulated controls.

To assess effects on vector titers, insulator A1 was introduced into thedouble-copy region of a third-generation lentiviral vector expressingGFP from a constitutive package promoter, and the viral titers and GFPexpression were measured. Insulator A1 did not affect adversely vectorGFP expression.

In the in vivo genotoxicity assay, a cell line transduced withgammaretroviral vectors produced tumors after transplantation in miceand allowed quantitation of genotoxic effects by measuring rates oftumor free survival. Effects of an insulator on genotoxicity werequantitated by the number of tumors formed in the mice and the rates oftumor free survival. Insulator A1 was inserted in the proximal portionof the 3′ LTR, from which it is copied into the 5′ LTR during reversetranscription and vector integration. The resulting topology placescopies of the insulator between the genomic regions located 5′ and 3′ ofthe integrated provirus and enhancer activity from the 5′ viral LTR andinternal Pgk promoter, but does not contain the enhancer in the 3′ LTR.This can decrease genotoxicity thus resulting in decreased tumorformation and increased survival of the animals. Gamma-retroviralreporter vectors flanked with insulator A1 or control regions were usedto transduce the growth factor-dependent cell line 32D, and 10independent sub-pools for each vector were transplanted into syngeneicC3H/HeJ mice. All 10 mice transplanted with mock-transduced cellsremained free of 32D cell-derived tumors, while nearly all micetransplanted with 32D cells transduced with vectors containing noinserts or a 790 bp neutral spacer developed tumors within a median of16 weeks (FIG. 5B). Flanking this vector with the cHS4 insulator delayedthe onset of tumor formation by several weeks, and reduced the frequencyof animals that developed tumors to 6 of 10. In contrast, only two of 10animals developed tumors following transplantation with 32D cellstransduced with the vector flanked with insulator A1 (FIG. 5B). Thefrequency of animals with tumors and the number of vector transductionevents in the original sub-pools suggested that flanking the vector withinsulator A1 reduced the overall rate of tumor formation 12-fold, from46.9 tumors per 10⁵ provirus to 3.9 tumors per 10⁵ provirus (FIG. 5C).In comparison, the cHS4 insulator reduced the overall rate of tumorformation 2.8-fold (to 16.9 tumors per 10⁵ provirus), while the neutralspacer had no statistically discernable effect on the rate of tumorformation. These results indicate that the discovered enhancer blockinginsulators can decrease substantially the risks of insertionalmutagenesis and genotoxicity.

Example 2: Characterization of Globin Vectors Comprising at Least OneInsulator

A presently disclosed expression cassette (designated as “ExpressionCassette 1”; as shown in FIG. 1), which comprises insulator A1, and ahuman β^(A)-globin gene encoding a threonine to glutamine mutation atcodon 87 (β^(A-T87Q)) operably linked to a β-globin LCR regioncomprising a HS2 region having the nucleotide sequence set forth in SEQID NO:9, a HS3 region having the nucleotide sequence set forth in SEQ IDNO:5, and a HS4 region having the nucleotide sequence set forth in SEQID NO:7, was generated. The rationale for using the variant β chain(β^(A)) is to facilitate the detection of the vector-encoded β-globingene, distinguishing it from endogenous or transfused beta chains. Theglutamine (GLN) residue at position 87 in the γ-globin chain augmentsthe anti-sickling activity of the gamma chain relative to the β chain,while preserving adult oxygen-binding characteristics of the β chain(Nagel et al. (1979)). In Vector 1, a point mutation altering codon 87(β^(A-T87Q), or β87) replaces the normal threonine with glutamine andaugments anti-sickling activity of the vector-encoded β chain. This β87chain has been safely used in a patient with HbE-thalassemia(Cavazzana-Calvo et al. (2010)).

Expression cassette 1 was incorporated or introduced to a lentivirusvector (designated as “Vector 1”). Vector 1 was introduced in bonemarrow cells of C57BL/6-Hbb th3/+ mice and transplanted to syngeneiclethally irradiated recipients as previously described (May et al.(2000), May et al. (2002), Lisowski et al. (2007)). The vector titer ofV1 was comparable to that of a lentivirus vector comprising anexpression cassette lacking insulator A1. The β-globin expression ofVector 1 was compared to that of a lentivirus vector (designated as“Vector 2”) comprising an expression cassette that lacks an insulatorand comprises a wild human β-globin gene operably linked to a β-globinLCR region comprising a HS2 region having the nucleotide sequence setforth in SEQ ID NO:9, a HS3 region having the nucleotide sequence setforth in SEQ ID NO:5, and a HS4 region having the nucleotide sequenceset forth in SEQ ID NO:6. In comparison to Vector 2, β-globin expressionof Vector 1 normalized to vector copy was equivalent or slightlyincreased, suggesting an added benefit for in vivo expression providedby the flanking barrier elements, as shown in FIG. 6.

Example 3: Evaluation of Enhancer Activity in Non-Erythroid K562 Cells

The enhancer activity of HS2 was evaluated in Non-erythroid K562 Cells.As shown in FIG. 7, GFP expression in K562 cells transduced with vectorsdriven by a minimal promoter linked to no enhancer (“Empty”, HS2, HS3-4,HS2-3-4 or the runx1 enhancer used as positive control (“RUNX1”).Background expression was on the order or 0.01% (“empty), but increasedover 10-fold with HS2-3-4 (“Lcr9”, 0.17%). This enhancement was mostlydue to HS2 (0.15%) but not HS3-4 (0.05%). All cell lines were comparablytransduced (mean vector copy number 2.5). The results support that HS2but not HS3-HS4 may pose an oncogenic risk in non-erythroidhematopoietic stem and progenitor cells.

Example 4: Novel Erythroid-Specific Enhancers

As shown in FIGS. 8 and 9, five erythroid-specific enhancers weresubstituted for HS2: ALAS Intron 1, ALAS Intron 8, BLVRB, PPDX, andSpectrin-alpha. The inventors have shown that all these five enhancersare powerful enhancers, and lack enhancer activity in non-erythroidtissues, and do not reduce the vector titer.

Example 5: Increasing Globin Lentiviral Vector Production Through 3′ LTRModifications

An essential feature of therapeutic globin vectors is to achieve a hightiter, sufficient for effective transduction of patient cells. By virtueof their large cargo, comprising a gene, promoter, enhancers and/or LCRelements, globin lentiviral vectors inherently have low titer,complicating their manufacture and limiting their clinical use. Thisproblem is further compounded by the incorporation of additional genomicelements such as an insulator, which further increase the size of thevector.

The inventors explored different modifications of the 3′ long terminalrepeat (LTR) of globin vectors to increase the titer of globin vectors.Over 62 variations were evaluated, numbered 1 through 62, modeled on alentivirus vector comprising a human β-globin gene operably linked to aβ-globin LCR region comprising a HS2 region having the nucleotidesequence set forth in SEQ ID NO:9, a HS3 region having the nucleotidesequence set forth in SEQ ID NO:5, and a HS4 region having thenucleotide sequence set forth in SEQ ID NO:7. In other words, all ofVectors #1 through Vector 62 comprise a β-globin LCR region comprising aHS2 region having the nucleotide sequence set forth in SEQ ID NO:9, aHS3 region having the nucleotide sequence set forth in SEQ ID NO:5, anda HS4 region having the nucleotide sequence set forth in SEQ ID NO:7.Vector #18 served as a baseline, comprising a standard U3 deletion inthe 3′LTR. Vector #1 (not depicted) comprised a full, i.e., wild-typeLTR, which cannot be used clinically. Modifications to the 3′LTR aredepicted in FIGS. 10A and 10B, and their titers shown in FIGS. 11 and 12(the Y axis shows the vector copy number of vector stocks manufacturedand tested under strictly identical conditions). Titrations weremeasured in triple replicas, performed in parallel by two operators, andrepeated in multiple experiments.

As shown in FIGS. 11 and 12, Vector #55 repeatedly showed a highertiter. This vector comprises a Woodchuck hepatitis post-regulatoryelement (WPRE) and a bovine growth hormone polyadenylation signal 3′ tothe R region in the 3′ LTR. The WPRE element is therefore nottransferred to the transduced cells.

The incorporation of these elements for enhancing the production ofglobin lentiviral vectors is essential to yield higher titers and hencefor the clinical usefulness of the vectors described in thisapplication.

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From the foregoing description, it will be apparent that variations andmodifications may be made to the presently disclosed subject matterdescribed herein to adopt it to various usages and conditions. Suchembodiments are also within the scope of the following claims.

All patents and publications and sequences referred to by accession orreference number mentioned in this specification are herein incorporatedby reference to the same extent as if each independent patent andpublication and sequence was specifically and individually indicated tobe incorporated by reference.

What is claimed is:
 1. An insulator comprising the CTCF binding sitesequence set forth in SEQ ID NO:18.
 2. The insulator of claim 1,comprising SEQ ID NO: 24 or SEQ ID NO:25.
 3. The insulator of claim 2,having the nucleotide sequence set forth in SEQ ID NO:1.
 4. Anexpression cassette comprising an insulator that comprises the CTCFbinding site sequence set forth in SEQ ID NO:18, and a globin gene or afunctional portion thereof operably linked to a β-globin locus controlregion (LCR) region.
 5. The expression cassette of claim 4, wherein theβ-globin LCR region does not comprise a Dnase I hypersensitive site-2(HS2) region.
 6. The expression cassette of claim 5, wherein theβ-globin LCR region does not comprise a core sequence of HS2.
 7. Theexpression cassette of claim 6, wherein the core sequence of HS2 has thenucleotide sequence set forth in SEQ ID NO:20 or SEQ ID NO:
 21. 8. Theexpression cassette of claim 4, wherein the β-globin LCR region does notcomprise a HS2 region that sustains the enhancer activity of HS2.
 9. Theexpression cassette of claim 4, wherein the β-globin LCR regioncomprises a Dnase I hypersensitive site-1 (HS1) region, a Dnase Ihypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4(HS4) region.
 10. The expression cassette of claim 9, wherein the HS3region is positioned between the HS1 region and the HS4 region.
 11. Theexpression cassette of claim 9, wherein the HS1 region is about 1.1 kbin length.
 12. The expression cassette of claim 11, wherein the HS1region has the nucleotide sequence set forth in SEQ ID NO:2.
 13. Theexpression cassette of claim 9, wherein the HS1 region is about 600 bpin length.
 14. The expression cassette of claim 13, wherein the HS1region has the nucleotide sequence set forth in SEQ ID NO:3.
 15. Theexpression cassette of claim 9, wherein the HS1 region is about 490 bpin length.
 16. The expression cassette of claim 15, wherein the HS1region has the nucleotide sequence set forth in SEQ ID NO:4.
 17. Theexpression cassette of claim 4, wherein the β-globin LCR region does notcomprise a HS1 region.
 18. The expression cassette of claim 18, whereinthe β-globin LCR region does not comprise a core sequence of HS1. 19.The expression cassette of claim 18, wherein the core sequence of HS1has the nucleotide sequence set forth in SEQ ID NO:22 or SEQ ID NO: 23.20. The expression cassette of claim 17, wherein the β-globin LCR regiondoes not comprise a HS1 region that sustains the function of HS1. 21.The expression cassette of claim 17, wherein the β-globin LCR regioncomprises a HS3 region and a HS4 region.
 22. The expression cassette ofclaim 21, wherein the HS3 region is positioned between the globin geneor functional portion thereof and the HS4 region.
 23. The expressioncassette of claim 9, wherein the HS3 region is about 1300 bp in length.24. The expression cassette of claim 23, wherein the HS3 region has thenucleotide sequence set forth in SEQ ID NO:5.
 25. The expressioncassette of claim 9, wherein the HS4 region is about 1.1 kb in length.26. The expression cassette of claim 25, wherein the HS4 region has thenucleotide sequence set forth in SEQ ID NO:6.
 27. The expressioncassette of claim 25, wherein the HS4 region has the nucleotide sequenceset forth in SEQ ID NO:7.
 28. The expression cassette of claim 9,wherein the HS4 region is about 450 bp in length.
 29. The expressioncassette of claim 28, wherein the HS4 region has the nucleotide sequenceset forth in SEQ ID NO:8.
 30. The expression cassette of claim 5,wherein the β-globin LCR region comprises a HS1 region having thenucleotide sequence set forth in SEQ ID NO:2, a HS3 region having thenucleotide sequence set forth in SEQ ID NO:5, and a HS4 region havingthe nucleotide sequence set forth in SEQ ID NO:6, and the β-globin LCRregion does not comprise a HS2 region.
 31. The expression cassette ofclaim 5, wherein the β-globin LCR region comprises a HS1 region havingthe nucleotide sequence set forth in SEQ ID NO:3, a HS3 region havingthe nucleotide sequence set forth in SEQ ID NO:5, and a HS4 regionhaving the nucleotide sequence set forth in SEQ ID NO:8, and theβ-globin LCR region does not comprise a HS2 region.
 32. The expressioncassette of claim 5, wherein the β-globin LCR region comprises a HS1region having the nucleotide sequence set forth in SEQ ID NO:4, a HS3region having the nucleotide sequence set forth in SEQ ID NO:5, and aHS4 region having the nucleotide sequence set forth in SEQ ID NO:8, andthe β-globin LCR region does not comprise a HS2 region.
 33. Theexpression cassette of claim 17, wherein the β-globin LCR regioncomprises a HS3 region having the nucleotide sequence set forth in SEQID NO:5 and a HS4 region having the nucleotide sequence set forth in SEQID NO:6, and the β-globin LCR region does not comprise a HS1 region or aHS2 region.
 34. The expression cassette of claim 4, wherein the β-globinLCR region comprises a HS2 region, a HS3 region, and a HS4 region. 35.The expression cassette of claim 34, wherein the HS2 region is about 860bp in length.
 36. The expression cassette of claim 35, wherein the HS2region has the nucleotide sequence set forth in SEQ ID NO:9.
 37. Theexpression cassette of claim 34, wherein the HS3 region is about 1300 bpin length.
 38. The expression cassette of claim 37, wherein the HS3region has the nucleotide sequence set forth in SEQ ID NO:5.
 39. Theexpression cassette of claim 34, wherein the HS4 region is about 1.1 kbin length.
 40. The expression cassette of claim 39, wherein the HS4region has the nucleotide sequence set forth in SEQ ID NO:7.
 41. Theexpression cassette of claim 34, wherein the β-globin LCR regioncomprises a HS2 region having the nucleotide sequence set forth in SEQID NO:9, a HS3 region having the nucleotide sequence set forth in SEQ IDNO:5, and a HS4 region having the nucleotide sequence set forth in SEQID NO:7.
 42. The expression cassette of claim 34, wherein the β-globinLCR region further comprises a HS1 region.
 43. The expression cassetteof claim 4, wherein the globin gene is selected from the groupconsisting of β-globin gene, γ-globin gene, and δ-globin gene.
 44. Theexpression cassette of claim 43, wherein the globin gene is humanβ-globin gene.
 45. The expression cassette of claim 44, wherein thehuman β-globin gene is selected from the group consisting of a wild-typehuman β-globin gene, a deleted human β-globin gene comprising one ormore deletions of intron sequences, and a mutated human β-globin geneencoding at least one anti-sickling amino acid residue.
 46. Theexpression cassette of claim 45, wherein the human β-globin gene ishuman β^(A)-globin gene encoding a threonine to glutamine mutation atcodon 87 (β^(3A-T87Q)).
 47. The expression cassette of claim 4,comprising one insulator having the nucleotide sequence set forth in SEQID NO:1.
 48. The expression cassette of claim 4, comprising two of theinsulator having the nucleotide sequence set forth in SEQ ID NO:1. 49.The expression cassette of claim 4, further comprising a β-globinpromoter.
 50. The expression cassette of claim 49, wherein the β-globinpromoter is positioned between the globin gene or functional portionthereof and the β-globin LCR region.
 51. The expression cassette ofclaim 49, wherein the β-globin promoter is a human β-globin promoterthat is about 613 bp in length.
 52. The expression cassette of claim 51,wherein the human β-globin promoter has the nucleotide sequence setforth in SEQ ID NO:10.
 53. The expression cassette of claim 49, whereinthe β-globin promoter is a human β-globin promoter that is about 265 bpin length.
 54. The expression cassette of claim 53, wherein the humanβ-globin promoter has the nucleotide sequence set forth in SEQ ID NO:11.55. The expression cassette of claim 4, further comprising a humanβ-globin 3′ enhancer.
 56. The expression cassette of claim 55, whereinthe human β-globin 3′ enhancer is positioned in the upstream of theglobin gene or functional portion thereof.
 57. The expression cassetteof claim 55, wherein the human β-globin 3′ enhancer is about 879 bp inlength.
 58. The expression cassette of claim 57, wherein the humanβ-globin 3′ enhancer has the nucleotide sequence set forth in SEQ IDNO:12.
 59. The expression cassette of claim 4, further comprising atleast one erythroid-specific enhancer.
 60. The expression cassette ofclaim 59, wherein the at least one erythroid-specific enhancer ispositioned between the globin gene or functional portion thereof andβ-globin LCR region.
 61. The expression cassette of claim 59, whereinthe at least one erythroid-specific enhancer has a nucleotide sequenceselected from the group consisting of SEQ ID NOS: 13, 14, 15, 16 and 17.62. The expression cassette of claim 59, comprising one, two or threeerythroid-specific enhancers.
 63. The expression cassette of claim 4,wherein the expression cassette allows for expression of the globin geneor functional portion thereof in a mammal.
 64. The expression cassetteof claim 63, wherein the expression cassette allows for expression ofhuman β-globin gene.
 65. The expression cassette of claim 62, whereinthe expression of the globin gene or functional portion thereof isrestricted to erythroid tissue.
 66. A recombinant vector comprising theexpression cassette of claim
 4. 67. The recombinant vector of claim 66,wherein the recombinant vector is a retroviral vector.
 68. Therecombinant vector of claim 67, wherein the retroviral vector is alentivirus vector.
 69. The recombinant vector of claim 66, wherein theexpression cassette comprises one insulator having the nucleotidesequence set forth in SEQ ID NO:1.
 70. The recombinant vector of claim66, further comprising one or both of a Woodchuck hepatitispost-regulatory element (WPRE) and a bovine growth hormonepolyadenylation signal in the 3′ long terminal repeat (LTR) of thevector.
 71. A non-naturally occurring or engineered nuclease comprisingthe expression cassette of claim
 4. 72. The nuclease of claim 7, whereinthe nuclease is selected from the group consisting of a non-naturallyoccurring or engineered zinc-finger nuclease (ZFN), a non-naturallyoccurring or engineered meganuclease, and a non-naturally occurring orengineered transcription activator-like effector nuclease (TALEN). 73.The nuclease of claim 71, wherein the nuclease comprises a DNA bindingdomain and a nuclease cleavage domain.
 74. The nuclease of claim 71,wherein the nuclease binds to a genomic safe harbor site.
 75. Thenuclease of claim 74, wherein the nuclease generates a double strandbreak (DSB) at the genomic safe harbor site.
 76. The nuclease of claim74, wherein the genomic safe harbor site is an extragenic genomic safeharbor site.
 77. The nuclease of claim 74, wherein the genomic safeharbor site is located on chromosome
 1. 78. The nuclease of claim 74,wherein the genomic safe harbor site meets all of the following fivecriteria: (1) distance of at least 50 kb from the 5′ end of any gene(e.g., from the 5′ end of the gene), (ii) distance of at least 300 kbfrom any cancer-related gene, (iii) within an open/accessible chromatinstructure (measured by DNA cleavage with natural or engineerednucleases), (iv) location outside a gene transcription unit and (v)location outside ultraconserved regions (UCRs), microRNA or longnon-coding RNA of the human genome.
 79. The nuclease of claim 71,wherein the expression cassette comprises two of the insulator havingthe nucleotide sequence set forth in SEQ ID NO:1.
 80. The nuclease ofclaim 71, which allows for targeted delivery of the expression cassette.81. A polynucleotide encoding the nuclease of claim
 71. 82. A vectorcomprising the polynucleotide of claim
 81. 83. The vector of claim 82,wherein the vector is a lentiviral vector.
 84. A non-naturally occurringor engineered CRISPR-Cas system comprising the expression cassette ofclaim
 4. 85. The system of claim 84, wherein the CRISPR-Cas systemcomprises a CRISPR-Cas nuclease and a single-guide RNA.
 86. The systemof claim 84, wherein the CRISPR-Cas system binds to a genomic safeharbor site.
 87. The system of claim 86, wherein the CRISPR-Cas systemgenerates a double strand break (DSB) at the genomic safe harbor site.88. The system of claim 85, wherein the genomic safe harbor site is anextragenic genomic safe harbor site.
 89. The system of claim 85, whereinthe genomic safe harbor site is located on chromosome
 1. 90. The systemof claim 85, wherein the genomic safe harbor site meets all of thefollowing five criteria: (1) distance of at least 50 kb from the 5′ endof any gene (e.g., from the 5′ end of the gene), (ii) distance of atleast 300 kb from any cancer-related gene, (iii) within anopen/accessible chromatin structure (measured by DNA cleavage withnatural or engineered nucleases), (iv) location outside a genetranscription unit and (v) location outside ultraconserved regions(UCRs), microRNA or long non-coding RNA of the human genome.
 91. Thesystem of claim 84, wherein the expression cassette comprises two of theinsulator having the nucleotide sequence set forth in SEQ ID NO:1. 92.The system of claim 84, which allows for targeted delivery of theexpression cassette.
 93. A polynucleotide encoding the CRISPR-Cas systemof claim
 84. 94. A vector comprising the polynucleotide of claim
 93. 95.The vector of claim 94, wherein the vector is a lentiviral vector.
 96. Acell transduced with the expression cassette of claim
 4. 97. A celltransduced with the recombinant vector of claim
 66. 98. A celltransduced with the nuclease of claim
 71. 99. A cell transduced with theCRISPR-Cas system of claim
 84. 100. The cell of claim 96, wherein thehematopoietic stem cell is a CD34⁺ hematopoietic stem cell.
 101. Apharmaceutical composition comprising an effective amount of the cell ofclaim 96 and a pharmaceutically acceptable carrier.
 102. A kit fortreating a hemoglobinopathy comprising the cell of claim
 96. 103. Amethod of treating a hemoglobinopathy in a subject, comprisingadministering an effective amount of the cell of claim 96 to thesubject, thereby enabling the subject's ability to produce red bloodcells containing normal hemoglobin.
 104. A method comprisingadministering an effective amount of the cell transduced with therecombinant vector of claim
 66. 105. The method of claim 104, whereinthe vector copy number of the recombinant vector in the cell thatprovides for the therapeutically relevant level of hemoglobin in thesubject is about 0.5-2 vector copy number per cell.
 106. The method ofclaim 103, wherein the method does not comprise administering animmunosuppressive agent.
 107. The method of claim 103, wherein thehemoglobinopathy is selected from the group consisting of hemoglobin Cdisease, hemoglobin sickle cell disease (SCD), sickle cell anemia,hereditary anemia, thalassemia, β-thalassemia, thalassemia major,thalassemia intermedia, α-thalassemia, and hemoglobin H disease. 108.The method of claim 103, wherein the subject is a human.
 109. The methodof claim 103, wherein the cell is from the subject.
 110. The method ofclaim 109, wherein the cell is from bone marrow of the subject.